1           General

1.1           Synthesis and characterization of iodixanol.

1. Priebe et al

Acta Radiologica Supplement, 399, 21-31 (1995)         

 

Iodixanol (Visipaque) is a new nonionic roentgen contrast medium intended for general use. Visipaque is a pharmaceutical formulation of iodixanol which is isotonic and isoosmotic with blood. Two synthetic routes from 5-nitro-isophthalic acid to iodixanol are described. The chemical structure is confirmed by spectroscopical data (¹H-NMR, ¹³C-NMR, electrospray-MS, UV, IR and Raman). Chromatographic characteristics are related to the isomerism of iodixanol.

 

1.2           Physical properties of iodixanol.

Eivindvik, K. and Sjøgren, E.

Acta Radiologica Supplement, 399, 32-38 (1995)

 

Iodixanol, the radiopaque in Visipaque, is a new nonionic, dimeric roentgen contrast medium for intravascular use. Compared to aqueous solutions of nonionic monomers, which have a higher osmolality than blood, aqueous solution of iodixanol have a lower osmolality due to the dimeric structure of the molecule. As a consequence of this advantageous property, solutions of all clinical concentrations of iodixanol can be made isotonic by the addition of salts of the key electrolytes sodium and calcium to the formulation. The viscosity of all iodixanol (Visipaque) solutions is less than or equal to that of iohexol (Nycodenz). Iodixanol itself is an amorphous and hygroscopic solid which is freely soluble in water. Partition coefficients show that iodixanol is even more hydrophilic than the nonionic monomers such as iohexol. The high hydrophilicity and the good aqueous solubility of iodixanol are due to the hydroxyl group in the dimer linkage and the hydrophilic amide side chains of the molecule.

 

1.3           Visipaque is isotonic to human and rat blood plasma.

Karlsson, J.O.G., Gregersen, M. and Refsum, H.

Acta Radiologica Supplement, 399, 39-42 (1995)

 

Even at its highest concentration, 320 mgI/ml, Visipaque - based on the nonionic dimer iodixanol - is isoosmotic to blood plasma, whereas Omnipaque (300 mgI/ml) - based on the nonionic monomer iohexol - has an osmolality of about twice that of the plasma. However, the fact that a solution is isoosmotic to plasma does not necessarily mean that it is isotonic to plasma. An isoosmotic solution can still cause a net movement of water over the plasma membranes of, for example, erythrocytes and endothelial cells.

 

Determination of the tonicity of Visipaque 320 mgI/ml and comparison with that of           Omnipaque 300 mgI/ml and hypertonic NaCl have been performed. No change in the water content of human erythrocytes was seen after mixing whole blood 10:1 with either Visipaque 320 mgI/ml or 155 mM NaCl, whereas a significant decrease in water content occurred with Omnipque 300 mgI/ml and 620 mM NaCl. No difference in the water content of rat erythrocytes was evident after mixing whole blood with Visipaque 320 mgI/ml or isotonic NaCl. However, as with human erythrocytes, a significant decrease in water content occurred after rat blood was mixed with Omnipaque 300 mgI/ml.

 

In conclusion, Visipaque 320 mgI/ml does not cause any net movement of water over the human or rat erythrocyte plasma membrane, i.e., Visipaque is isotonic to human and rat blood plasma.

 

1.4           Formulation, stability and compatibility of iodixanol.

Aars, E.V. and Eivindvik, K.

Acta Radiologica Supplement, 399, 50-58 (1995)

 

Iodixanol (Visipaque) is an isotonic, electrolyte-balanced roentgen contrast medium for intravascular use. The patented and well-proven formulation and the rationale for it are described, and the efficacy and safety documented. The stability of iodixanol is well within the specifications under all relevant conditions, both in glass and polypropylene bottles; the product has a recommended shelf-life of at least 36 months when stored at room temperature and protected from light. Heating to body temperature before use is acceptable and recommendable, and storage at 37⁰C for 1 month does not jeopardize product quality. Iodixanol has no apparent immediate in vitro incompatibility reactions with drugs used in connection with roentgen contrast examination.

1.5           Preclinical pharmacokinetics and general toxicology of iodixanol.

Heglund, F. et al

Acta Radiologica Supplement, 399, 69-82  (1995)

 

To document the safety of iodixanol and to assess its pharmacokinetic properties, extensive tests have been performed. Iodixanol was rapidly excreted, mainly via the     kidney, with a plasma half-life in rats and monkeys of 25 and 76 min. respectively. The pharmacokinetic data was consistent with an extracellular distribution of iodixanol. During the 24 hours post-dosing, the urinary excretion was from 72 to 100% in rats and 78% in monkeys. Biliary excretion was 1.5% during the first 4 hours in rats. Fecal excretion was about 7% in rats and 0.8% in monkeys over the first 24 hours after injection. Approximately 0.5 and 1% of the dose was found in the kidneys of rats and monkeys, respectively, 24 hours after dosing. Acute toxicity of iodixanol in rats was low, with an LD₅₀ greater than 21gI/kg. In mice the LD₅₀ was 21gI/kg and the approximated median lethal dose (ALD₅₀) was found to range from 15 to 21 gI/kg. A single dose of 1 and 3 gI/kg was well tolerated in monkeys. As for other roentgen contrast media, a reversible, dose-related, vacuolation of the proximal tubules in the kidneys was seen in the acute and subacute studies in rats and monkeys. No relationship was seen between the vacuolisation and kidney function. Local tolerance studies demonstrated a low irritation potential for iodixanol when injected by a variety of intravascular and extravascular routes.

 

The reproductive capacity of male and female rats was unaffected by iodixanol when administered daily at doses up to 2 gI/kg/day. No teratogenic potential in rats and rabbits of iodixanol was observed. Further, no toxic effects on pups were seen when rats were dosed during the lactation period. Each of 4 standard genotoxicity tests was negative. No antigenic potential of iodixanol was observed when assessed by the passive cutaneous anaphylaxis test and the active systemic anaphylaxis test in guinea pigs.

 

The intravascular tolerability of iodixanol is high and, therefore, iodixanol should be considered as a safe roentgen contrast medium for intravascular use.

 

1.6           Iodixanol: A nonionic iso-osmotic centrifugation medium for the formation of self generated gradients.

Ford, T., Graham, J. and Rickwood, D.

Anal. Biochem., 220, 360-366 (1994)

 

The physical and biological properties of iodixanol, a new nonionic density gradient medium, are described in this paper. It is effectively a dimer of Nycodenz and it exhibits two significant advantages over previous iodinated density gradient media- its  aqueous solutions are iso-osmotic up to a density of 1.32 g/ml and it is capable of forming self-generating gradients in 1 to 3 h. It has a very low toxicity towards biological material and enzyme assays can be carried out in its presence.

 

1.7           Spectrophotometric determination of iodixanol in subcellular fractions of mammalian cells.

Schroder, M., Schafer, R. and Friedl, P.

Anal. Biochem., 244, 174-176 (1997)

 

Nonionic iodinated density gradient media are widely used for the separation of cells and organelles. The most suitable of them is iodixanol. It displays no cytotoxicity and a number of marker enzymes for cellular organelles can be assayed in the presence of iodixanol. In contrast to other nonionic iodinated density gradient media, such as metrizamide or Nycodenz, iodixanol readily forms self-generated gradients, thus obviating the preparation of gradients using a gradient marker.

 

A subcellular fractionation of cellular organelles is evaluated by the distribution of marker enzymes in the gradient and the density profile of the gradient. The density profile of a gradient is commonly analyzed by measuring the refractive index of the gradient fractions. Cellular material present in subcellular fractions interferes with the determination of the refractive index, thus necessitating a mock fractionation to determine the density profile of the gradient.

 

Here we report the direct and specific determination of iodixanol as an example for a nonionic density gradient medium in subcellular fractions. No mock runs are necessary to determine the density profile of the gradient when this method is used. This assay should be more convenient for many laboratories because a refractometer is not required.

 

1.8           Determination of particle sedimentation rate by ultrasonic interferometry: role of particle size, density and volume fraction

Razavian, S.M., Wenby, R.B., Fisher, T.C. and Meiselman, H.J.

Biorheology, 34(4/5), 349-362 (1997)

 

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25°C using latex spheres of different diameters (7 to 20 mm) and densities (1.062 to 1.190 g/cm³) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.

 

1.9           Iodixanol is readily eliminated by hemodialysis

Berg, K.J., Rolfsen, B. and Stake, G.

Acta Radiologica, 39, 372-374 (1998)

 

The dialyzability of the high-molecular X-ray contrast medium iodixanol was examined in an in vitro hemodialysis model using two different hollow fiber membranes: one high flux (polysulfone) membrane and one intermediate-flux (cellulose triacetate) membrane. Blood flow was 200 ml/min and membrane area 1.3 m². The dialyzer clearance of iodixanol dissolved in a mixture of leucocyte-filtered SAG-M blood and compatible citrate plasma was 143.2 ± 3.6 ml/min for the polysulfone membrane and 113.0 ± 3.6 ml/min for the triacetate membrane. Iodixanol is readily dialyzed through commercial high-flux membranes.

 

1.10           Stability of the X-ray contrast agent iodixanol - 3,3’5,5’-tetrakis(2,3dihydroxy-propylcarbamoyl)-2,2’,4,4’,6,6’-hexaiodo-N,N’-(2-hydroxypropane-1,3-diyl)-diacetanilide towards acid, base, oxygen, heat and light.

Priebe, H. et al.

1. Clin. Pharm. and Therapeut., 24, 227-235 (1999)

 

Background: During the production of the X-ray contrast agent iodixanol the drug substance may be exposed to acid, base, air, heat and daylight, conditions that may cause decomposition products.

 

Objectives: To investigate the chemical stability of iodixanol under accelerating conditions.

 

Method: Chemometrical stability studies were undertaken to investigate the effect of acid and base on the contrast agent’s stability.

 

Results: Cleavage of the central bridge in iodixanol occurred under ultraviolet irradiation via a Norrish Type-II reaction. Basic conditions (pH 14) combined with heat (60°C) initiated a cyclization reaction. Less than 1% iodixanol decomposed in solution heated to 140°C for 2 days or under both basic conditions (pH 11, 20°C, 5 days) and acidic conditions (pH 0-4, 80°C, 5 days) or under oxygen atmosphere (100°C, 3 days).

 

Conclusion: Even under highly acidic and basic conditions, iodixanol is stable.

2           Macromolecules

 

2.1           A new method for the rapid separation of plasma lipoproteins

Graham, J.M., Higgins, J.A. and Gillot, T.

Atherosclerosis, 115 (Suppl), S123 (1995)

 

Variations in the relative amounts of plasma lipoprotein classes and /or molecular changes in their lipid components and apoproteins, are important factors in the development of atherosclerosis. A new equilibrium centrifugation method, which avoids high salt gradients, provides high resolution and concentration of lipoproteins for electrophoretic, compositional and structural analysis. The method uses a new non-ionic medium, iodixanol, available as a 60% (w/v) solution called OptiPrep (Axis-Shield PoC, Oslo). Plasma is adjusted to 12.5% iodixanol; overlaid with 0.1-0.2x its volume of saline and centrifuged at approx. 350,000g for 2.5-3 h in a vertical or near-vertical rotor. Using 3.9 ml tubes, unloaded dense end first; soluble proteins are contained in the first 1.0 ml, HDL in the 1.2-2.4 ml fraction; LDL in the 2.6-3.4 ml fraction and VLDL in the 3.6-3.9 ml fraction. There is little overlap between the three classes. Fractions can be analysed directly, by gel electrophoresis and for lipid content. The method may resolve different sub-classes of these lipoproteins: this is currently being investigated. Because of the concentration effect achieved by the gradient, Lp(a), which is recovered in a single 0.25 ml fraction, can be easily demonstrated on gels when levels in the whole plasma are below the limits of detection. The technique thus provides a means of fractionating and concentrating all plasma lipoproteins in a single step; it should prove to be a valuable diagnostic and analytical tool in studies of lipoprotein metabolism.

 

2.2           A novel method for the rapid separation of plasma lipoproteins using self-generated gradients of iodixanol.

Graham, J. et al

Atherosclerosis, 124, 125-135 (1996)

 

We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new

is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionation. Because the medium is inert, fractions from the gradient can be analyzed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classes may occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research and clinical laboratory.

 

2.3           Isolation of plasmid DNA.

Rickwood, D.and Patel, N.

Mol. Biol. Cell, 7, 162a (1996)

 

Centrifugation of DNA in CsCl-ethidium bromide gradients remains a widely used technique for the isolation of high purity plasmid DNA. There are two major problems with separations using this technique. The first is that because ethidium bromide intercalates into the DNA helix, it is a powerful mutagen, leading to dangers in working with it and difficulties in disposing of the ethidium bromide after use. The other problem is that ethanol precipitation of the plasmid can cause the precipitation of CsCl thus complicating recovery of the DNA. We have devised a new method for the purification of plasmid DNA using self-generating OptiPrep gradients containing a fluorescent dye, DAPI, to mark the position of the DNA in the gradient. Crude plasmid DNA is prepared using the standard alkaline lysis method and then purified by centrifugation in a gradient of 28% OptiPrep containing 0.005% DAPI at 150,000g for 40 hours at 5°C. The native DNA is separated from the denatured chromosomal DNA, RNA and proteins. As judged by electrophoresis, the plasmid DNA obtained after centrifugation is free of contaminating chromosomal DNA and RNA and can be ethanol precipitated directly from the gradient solution.

 

2.4           Rate zonal sedimentation of proteins in one hour or less.

Basi, N.S. and Rebois, V.

Anal. Biochem., 251, 103-109 (1997)

 

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 ul) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of s_20,w  versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated a-subunits were determined. The results were similar to those obtained in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

 

2.5           Effect of dietary fish oil or sunflower oil on plasma lipoproteins and hepatic gene expression in the hamster

Bennett, A.J., Kendrick, J.S., Anderton, K.L., Higgins, J.A. and White, D.A.

Atherosclerosis, 130 (Suppl. 1), S24 (1997)

 

An increased dietary consumption of fish oils reduces the occurrence of coronary artery disease. Although this probably involves several mechanisms, one effect of increased consumption of fish oil is a fall in plasma triacylglycerol (TAG) in humans and experimental animals. This has led to the idea that fish oil fatty acids lower VLDL secretion by the liver and hence reduce the risk of atherosclerosis. To investigate this, we have supplemented the diets of hamsters with 10% fish-oil or 10% sunflower oil and examined blood lipoproteins, VLDL secretion and the mRNA levels of genes involved in apolipoprotein B metabolism. Hamsters were fed 1.5 ml of either fish oil (Maxepa) or sunflower oil by daily gavage. Blood samples were taken at weekly intervals. The lipids were analysed and the lipoprotein classes separated on iodixanol gradients and by agarose electrophoresis. In fish oil fed animals, plasma TAG fell by 25% over a three-week period. There was no significant change in the TAG levels of either chow fed animals or sunflower oil fed animals or in the cholesterol levels of the three groups of animals. Plasma VLDL were considerably reduced and there was a small increase in LDL and HDL in the fish-oil fed animals. IN the sunflower oil fed and chow fed animals, there was no significant change in the plasma lipoproteins. Consistent with these results from in vivo experiments, secretion of VLDL by hepatocytes isolated from fish-oil hamsters were considerably reduced compared with hepatocytes from chow-fed and sunflower-oil fed animals. The fall in plasma VLDL in fish-oil fed hamsters was not accompanied by a fall in LDL. To determine whether this is due decreased LDL uptake we determined the mRNA levels for the LDL receptor (LDL-R) in liver from hamsters fed fish-oils, sunflower oil or chow for three weeks using the mRNA protection assay. The mRNA for apo-B, HMG-CoA reductase and microsomal triacylglycerol transfer protein (MTP) were also measured in the same liver samples to examine possible effects on other candidate proteins involved in apo-B metabolism 9Table 1). Dietary fish-oils reduced the levels of mRNA for the LDL-R by approximately 60% while dietary sunflower oil had no significant effect, mRNA for HMG-CoA reductase was reduced 60% by feeding fish-oils and 20% by feeding sunflower oils. These results suggest that there may be a complex metabolic relationship between apo-B synthesis and uptake by the liver which is perturbed by feeding fish-oils.

 

2.6           Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver

Wilkinson J., Higgins, J.A., Fitzsimmons, C. and Bowyer, D.E.

Arterioscler. Thromb. Vasc. Biol., 18: 1490-1497 (1998)

 

Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low-density lipoprotein (VLDL), increased low-density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by >70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of r~1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of r<l .21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus down-regulating the expression of the LDL receptor.

 

2.7           Carotenoid composition and antioxidant potential in subfractions of human low-density lipoprotein.

Lowe, G.M. et al

Ann. Clin. Biochem., 36, 323-332 (1999)

 

Carotenoids and vitamin E are transported in human plasma complexed with lipoproteins. The bulk of them are associated with low-density lipoprotein (LDL) where they may act as antioxidants and thus delay the onset of atherosclerosis. In this study we have used a simple, rapid ultracentrifugation technique in which plasma lipoproteins are fractionated in self-generated gradients of iodixanol, a non-ionic iodinated density gradient medium. The carotenoid content and composition of a number of LDL subfractions was determined by reversed-phase HPLC. Lycopene, b-carotene and b-cryptoxanthin were mainly located in the larger, less-dense, LDL particles whereas lutein and zeaxanthin were found preferentially in the smaller, more-dense, LDL particles. When the antioxidant content of these fractions was expressed per mg of LDL protein, a different distribution was found with significantly lower concentrations of carotenoid and vitamin E associated with the smaller protein-rich, fractions of LDL. Strong positive correlations were found for total carotenoid and vitamin E concentrations with the lag-time of Cu²⁺-mediated oxidation of LDL subfractions. The more dense LDL subfractions, which had lower levels of these antioxidants, were more readily oxidised, highlighting their possible role in atherosclerotic events.

 

 

2.8           Blood appearance, metabolic transformation and plasma transport proteins of ¹⁴C-   astaxanthin in Atlantic salmon (Salmo salar L.)

Aas, G.H., Bjerkeng, B., Storebakken, T. And Ruyter, B.

Fish Physiol. Biochem., 21, 325-334 (1999)

 

The time of appearance in blood, and transport of astaxanthin, and catabolic transformation of astaxanthin to idoxanthin were investigated in Atlantic salmon (Salmo salar) that had been force-fed a single dose ¹⁴C-astaxanthin. In addition to the LPs, a major protein, associated with radiolabeled astaxanthin was detected. The maximum level of radiolabeled carotenoids in blood was attained 30 h after administration of ¹⁴C-astaxanthin. Radioactive idoxanthin (combined 3’4’-cis and 3’4’-trans glycolic isomers of idoxanthin) appeared after 6 h and a stable level was obtained after 18 h. LPDP and LP, separated by ultracentrifugation, contained on average 89 and 11% of the total radioactivity in plasma, respectively. During the 168 h experiment, maximum radioactivity in LP appeared after 22h. Separation of plasma by ultracentrifugation on a discontinuous NaCl/KBr-gradient and an iodixanol-gradient confirmed that most of the radiolabel carotenoids were present in the HDPF that did not contain LPs (58%), whereas HDL and LDL contained 36 and 6% of the radioactivity, respectively. Of the recovered radioactivity, astaxanthin in the HDPF comprised 82%, idoxanthin 5% and unidentified compounds 12%, whereas HDL contained 78% astaxanthin, 22% idoxanthin and no unidentified compounds. Protein from the fractions with high density and high radioactivity (iodixanol-gradient) were separated by PAGE under non-denaturing conditions and showed a radioactive band with parallel migration length to BSA and salmon albumin. These results show that astaxanthin is rapidly converted to idoxanthin and that the majority of astaxanthin in the plasma is associated with a protein other than LPs, presumably albumin. The identity of this protein requires verification.

 

2.9           In healthy adults, postprandial lipaemia results in triglyceride enrichment of very low-density lipoprotein, enhanced oxidative stress and deterioration in endothelial function

Anderson, R.A. et al

Atherosclerosis Suppl 154(2), 434 (1999)

 

There is a significant relationship between postprandial lipaemia (PPL) and coronary artery disease in patients with both normal and abnormal carbohydrate metabolism. PPL has recently been shown to cause endothelial dysfu8nction in normal individuals: attenuated by antioxidants. We therefore studied the effects of PPL on endothelial function (EF), free radicals (FR) and triglyceride (TG) metabolism in healthy subjects. Methods: Twelve subjects with no history of vascular disease or diabetes were studied, (five male, seven female; mean age 43 years). After a 12-h overnight fast, EF, lipid profiles and venous FR levels were measured in the fasting and peak postprandial phase (at 4 h) subsequent to ingestion of a standard fat meal. Flow-mediated dilatation (FMD) was assessed, as a measure of EF in the brachial artery, and expressed as the percentage change in brachial artery diameter. Lipoprotein subfractions were assessed using a density gradient medium, iodixanol. Lipid-derived FR levels were measured in venous blood by electron paramagnetic resonance spectroscopy; results expressed in arbitrary units. Results: There was an increase in venous FR postprandially (mean ± SD), 2.4 ± 0.1 to 3.3 ± 0.2 (P < 0.05), and a decrease in FMD, 6.26 ± 1.3% to 4.8 ± 1.3% (P < 0.05). TG levels (in mmol/l) were also significantly elevated at 4 h., 1.3 ± 0.5 to 2.2 ± 0.7 (P < 0.05). There were no changes in TG content of high- and low-density lipoprotein cholesterol but there was an increase in TG content of very low-density lipoprotein (VLDL), 54 ± 6.5% to 59.3 ± 5.7%. Conclusion: We show for the first time that PPL in healthy individuals is associated with increased lipid-derived free radicals and deterioration of endothelial function. This was associated with relative TG-enrichment of VLDL particles.

 

2.10           Ciprofibrate reduces the postprandial generation of triglyceride-rich lipoproteins and attenuates the associated endothelial dysfunction and oxidative stress in non-insulin dependent diabetes mellitus

Evans, L.M. et al

Atherosclerosis Suppl 154(2), 434 (1999)

 

Introduction: Triglyceride-rich lipoproteins (TGRL) may be involved in athero­genesis by mechanisms including endothelial dysfunction and enhanced oxida­tive stress. Non-insulin-dependent diabetes mellitus (NIDDM) results in excess vascular disease and exaggerated excursions of postprandial TGRL. We studied the effect of fibrate therapy on the relationship between postprandial lipaemia (PPL), endothelial function (EF) and oxidative stress in NIDDM. Methods: Twenty NIDDM patients were studied following an overnight fast and 4 h after a fatty meal. Lipoproteins were separated using an iodixanol density gradient medium. Free radicals (FR) were directly measured using electron paramag­netic resonance spectroscopy. EF was assessed by measuring flow-mediated brachial artery dilatation (FMD). Subjects were randomised in a double-blind manner to 3 months of placebo (P) or ciprofibrate (C) (100 mg OD). Results (mean ± SD): Seventeen subjects completed the study. At base line, both groups exhibited similar changes in FMD [(% change) 3.8 ± 1.8% to 1.8 ± 1.3% (C) vs. 3.3 ± 1.7 to 1.7 ± 1.1% (P)]. Increase in FR [(arbitrary units) 2.9 ± 1.3 (C) vs. 3.1 ± 1.5 (P)]. Rise in plasma TG [(mmol/l) 2.8 ± 2.1 to 6.7 ± 6 (C) vs. 2.8 ± 1.7 to 7 ± 7.3 (P)] and TG enrichment of very low-density lipoprotein (VLDL) [(% TG content) 59.6 ± 4.6% to 73.4 ± 6.9% (C) vs. 61.2 ± 5.9% to 76.1 ± 9.8% (P)]. Following treatment fasting and PP, FMD improved in the treatment group with reductions in fasting and PP TG, VLDL-TG content and FR. FMD, 4.8 ± 1.1 to 3.4 ± 1.1% (C) vs. 3.4 ± 1.2 to 1.8 ± 1.1% (P) (P <0.05). TG, 1.5 ± 0.8 to 2.8 ± 1.3 mmol/l (C) vs. 3.1 ± 2.1 to 6.6 ±4.1(P) mmol/l (P< 0.05). VLDL-TG (%), 50.1 ± 6.2 to 59.5 ± 4.3% (C) vs. 60.6 ± 3.9 to 72.9 ± 6.9% (P) (P<0.05). FR, 0.3 ± 0.6 (C) vs. 1.1 ± 0.9 (P) (P<0.05). Conclusions: This study demonstrates that ciprofibrate may reduce the risk of vascular disease in NIDDM by mechanisms involving improved EF, attenuated PPL, enhanced catabolism of TGRL and reduced FR release.

 

2.11           Consequences of interaction of a lipophilic endotoxin antagonist with plasma lipoproteins

Rose, J.R. et al

Antimicrobial Agents and Chemotherapy, 44(3), 504-510 (2000)

 

E5531, a novel synthetic lipid A analogue, antagonizes the toxic effects of lipopolysaccharide, making it a potential intravenously administered therapeutic agent for the treatment of sepsis. This report describes the distribution of E5531 in human blood and its activity when it is associated with different lipoprotein subclasses.  After in vitro incubation of  (¹⁴C) E5531 with blood, the great majority (>92%) of material was found in the plasma fraction.  Analysis by size-exclusion and affinity chromatography and density gradient centrifugation indicates that (¹⁴C)E5531 binds to lipoproteins, primarily high-density lipoproteins (HDLs), with distribution into low-density lipoproteins (LDLs) and very low density lipoproteins (VLDLs) being dependent on the plasma LDL or VLDL cholesterol concentration.  Similar results were also seen in a limited study of (¹⁴C)E5531 administration to human volunteers.  The potency of E5531 in freshly drawn human blood directly correlates to increasing LDL cholesterol levels.  Finally, preincubation of E5531 with plasma or purified lipoproteins indicated that binding to HDL resulted in a time-dependent loss of drug activity.  This loss in activity was not observed with drug binding to LDLs or to VLDLs or chylomicrons.  Taken together, these results indicate that E5531 binds to plasma lipoproteins, making its long-term antagonistic potency dependent on the plasma lipoprotein composition.

 

 

2.12           Ciprofibrate therapy improves endothelial function and reduces postprandial lipemia and oxidative stress in type 2 diabetes mellitus

Evans, M. et al

Circulation, 101, 1773-1779 (2000)

 

Background-Exaggerated postprandial lipemia (PPL) is a factor in atherogenesis, involving endothelial dysfunction and enhanced oxidative stress.  We examined the effect of ciprofibrate therapy on these parameters in type 2 diabetes mellitus.

 

Methods and Results-Twenty patients entered a 3-month, double blind, placebo-controlled study.  Each subject was studied fasting and after a fatty meal, at baseline, and after 3 months of treatment.  Glucose and lipid profiles were measured over an 8-hour postprandial period.  Endothelial function (flow-mediated endothelium-dependent vasodilatation [FMDI) and oxidative stress (electron paramagnetic resonance spectroscopy) were measured after fasting and 4 hours postprandially.  At baseline, both groups exhibited similar PPL and deterioration in endothelial function.  After ciprofibrate, fasting and postprandial FMD values were significantly higher (from 3.8±1.8% and 1.8 ±1.3% to 4.8 ± 1.1% and 3.4 ± 1.1%; P<0.05). This was mirrored by a fall in fasting and postprandial triglycerides (3.1±2.1 and 6.6±4.1 mmol/L to 1.5±0.8 and 2.8±1.3 mmol/L, P<0.05). Fasting and postprandial HDL cholesterol was also elevated (0.9±0.1 and 0.8±0.1 mmol/L and 1.2±0.2 and 1.2±0.1 mmol/L, P<0.05). There were no changes in total or LDL cholesterol.  Fasting and postprandial triglyceride enrichment of all lipoproteins was attenuated, with cholesterol depletion of VLDL and enrichment of HDL.  There were similar postprandial increases in oxidative stress in both groups at baseline, which was significantly attenuated by ciprofibrate (0.3±0.6 versus 1.5± 1.1 U, P<0.05).

 

Conclusions-This study demonstrates that fibrate therapy improves fasting and postprandial endothelial function in type 2 diabetes.  Attenuation of PPL and the associated oxidative stress, with increased HDL cholesterol levels, may be important.

 

2.13           Collagen-bound von Willebrand factor has reduced affinity for factor VIII

Bendetowicz, A.V., Wise, R.J. and Gilbert, G.E.

J. Biol. Chem., 274(18), 12300-12307 (2000)

 

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma.  Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied.  We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma.  Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites.  In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with K, increasing from 0.9 to 3.8 nm.  However, one factor VIII-binding site remained available on each vWf subunit.  A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen.  In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf.  We conclude that Collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

 

2.14           Plasma appearance and distribution of astaxanthin E/Z and R/S isomers in plasma lipoproteins of men after single dose administration of astaxanthin

Osterlie, M., Bjerkeng, B. And Liaaen-Jensen, S.

1. Nutr. Biochem., 11, 482-490 (2000)

 

Appearance, pharmacokinetics, and distribution of astaxanthin E/Z and R/S isomers in plasma and lipoprotein fractions were studied in 3 middle-aged male volunteers (37-43 years) after ingestion of a single meal containing a 100 mg dose of astaxanthin. The astaxanthin source consisted of 74% all-E-, 9% 9Z-, 17% 13Z-astaxanthin (3R, 3’R, 3R, 3’S: meso-, and 3S, 3’S-astaxanthin in a 1:2:1 ratio). The plasma astaxanthin concentration – time curves were measured during 72 hr. Maximum levels of astaxanthin (1.3 ± 0.1 mg/L) were reached 6.7 ± 1.2 hr after administration, and the plasma astaxanthin elimination half-life was 21 ± 11 hr. 13Z-astaxanthin accumulated selectively, whereas the 3 and 3’R/S astaxanthin distribution was similar to that of the experimental meal. Astaxanthin was present mainly in very low-density lipoproteins containing chylomicrons (VLDL/CM); 36-64% of total astaxanthin), whereas low-density lipoprotein (LDL) and high-density lipoprotein (HDL) contained 29% and 24% of total astaxanthin, respectively. The astaxanthin isomer distribution in plasma, VLDL/CM, LDL and HDL was not affected by time. The results indicate that a selective process increases the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin during blood uptake and that astaxanthin E/Z isomers have similar pharmacokinetics.

 

2.15           Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of b-amyloid peptide

Ward, R.V. et al

Biochem. J., 348, 137-144 (2000)

 

The b-amyloid (Ab) peptide, a major component of senile plaques in Alzheimer disease brain, has been shown previously to undergo a process of polymerisation to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Ab responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Ab peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Ab were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions were determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillary structures, but not to monomeric peptide or sheet-like structures of polymerized Ab. Biological Activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillary Ab; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Ab on cell viability is not confined to a single conformational form but that both fibrillary and protofibrillar species have the potential to be active in this assay.

 

2.16           Capillary isotachophoretic analysis of serum lipoprotein using a carrier ampholyte as spacer ion

Inano, K., Tezuka, S., Miida, T. and Okada, M.

Ann. Clin. Biochem., 37, 708-716 (2000)

 

We have developed a novel analytical method for serum lipoproteins using a commercially available capillary electrophoresis apparatus, BioFocus 3000 (Bio-Rad Laboratories Co Ltd, USA). The analytical principle is isotachophoresis (ITP), using a carrier ampholyte, BioLyte 7/9, as a spacer ion. The method allows a much higher resolution of lipoproteins than of amino acid mixtures. Serum lipoproteins are normally separated into 13-15 peaks, including some shoulder peaks. The reproducibility of repeated analysis within a day was relatively good with the coefficient of variation within the range 0.9-1.1%. VLDL, LDL and HDL prepared by discontinuous density ultracentrifugation could be further separated by capillary ITP. This high-resolving ability of our method enabled detection of small amounts of abnormal lipoprotein species. For example, small dense LDL, which is thought to be an atherogenic lipoprotein, could be detected within the LDL group peak. Moreover, an abnormal HDL, apolipoprotein E-rich HDL, was also detected by a single analysis. These findings suggest that our capillary ITP method is a useful means for detailed analysis of lipoproteins and thus for clinical diagnosis of hyperlipoproteinaemic subjects.

 

2.17           A randomized trial of the effects of garlic oil upon coronary heart disease risk factors in trained male runners

Zhang, X-H., Lowe, D., Giles, P., Fell, S., Board, A. R., Baughan, J. A., Connock, M. J. and Maslin, D. J.

Blood Coagulat. Fibrinolysis, 11, 67-74 (2000)

 

Most trials of bulb garlic and garlic powder tablets indicate reduced coronary heart disease (CHD) risk in elevated-risk subjects. Most persons taking garlic supplements lack overt risk of Cl-ED. However, no trials have tested steam-distilled garlic oil (GO) capsules with healthy subjects. The objectives of the present randomized, double-blind, placebo-controlled study were to determine whether GO capsules reduce CHD risk in trained male runners. Twenty-seven volunteers (mean age, 28.8 years) completed the study. Each took 12.3 mg/day GO (or placebo) capsules for 16 weeks. Main outcome measures were 95% confidence intervals (CIs) between GO and placebo groups for differences in changes of blood pressure (BP), plasma lipids, total antioxidant status (TAS), low-density lipoprotein (LDL) composition and blood clotting factors. Principal results as mean differences (95% CI) between GO and placebo are: pulse, 2.9 beats/mm (-0.8 to 6.7), P = 0.12; systolic BP, -4.5 mmHg (-10.8 to 1.9), P = 0.16; plasma total cholesterol, 0.01 mmol/l (-0.34 to 0.37), P = 0.95; plasma triglycerides, -0.20 mmol/l (-0.43 to 0.03), P = 0.09; plasma TAS, 45 pmol Trolox equivalent/l (-35 to 124), P = 0.26; LDL density, 0.0019 g/ml (-0.0005 to -0.0043), P = 0.12; LDL triglycerides/protein, -0.078 mg/mg (-0.149 to -0.007), P = 0.03; LDL cholesterol/protein, 0.24 mg/mg (-0.69 to 0.22), P = 0.3; LDL TAS/triglycerides, 29 nmol/mg (11 to 68), P = 0.15; prothrombin time, 0.99s (-0.36 to 2.35), P = 0.14; partial thromboplastin time, 3.0s (-1.0 to 7.1), P = 0.13. Results were null statistically. Trends with GO were mostly towards lower CHD risk, and a larger study (-150 subjects) is required to test their validity.

 

2.18           Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors

Brügger, B. et al

EMBO J., 19(6), 1272-1278 (2000)

 

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoyl-phosphatidylserine (DOPS) and 1,2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999).  The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes.  We can confirm that isolated NSF can fuse liposomes of the specified composition.  However, this activity of NSF is resistant to inactivation of- N-ethylmaleimide and does not depend on the presence of a-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either a-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase or even 3 mM magnesium ions can also cause lipid mixing.  In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids.  A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes.  A variety of minor perturbations could then trigger coalescence.

 

 

2.19           The relationships between post-prandial lipaemia, endothelial function and oxidative stress in healthy individuals and patients with type 2 diabetes

Anderson, R.A. et al

Atherosclerosis, 154, 475-483 (2001)

 

Post-prandial lipaemia (PPL) is a factor in atherosclerosis and results in reversible endothelial dysfunction in healthy individuals. Oxidative stress and triglyceride (TG)-rich lipoproteins have been implicated. Type 2 diabetes (NIDDM) results in exaggerated PPL. We attempted to delineate the mechanisms of PPL induced, endothelial dysfunction (EF) and oxidative stress in 12 NIDDM and 12 matched healthy subjects. Subjects underwent a fat tolerance test, with endothelial function assessed by flow-mediated vasodilatation and oxidative stress measured by venous lipid-derived free radicals ex vivo and lipid peroxidation products over the postprandial phase. Fasting TG, post-prandial hypertriglyceridaemia and the TG enrichment of all lipoproteins was significantly greater in NIDDM. Post-prandial endothelial function inversely correlated with fasting HDL-C (r = -0.84, P= 0.001) in both the control and NIDDM groups. The deterioration in EF in the NIDDM group also correlated with TG enrichment of VLDL and LDL. PPL in both groups also resulted in increased oxidative stress. The increment in free radicals correlated with TG enrichment of VLDL in both groups and was, therefore, greater in NIDDM. Thus, PPL – with the production of TG-enrichment of VLDL – results in endothelial dysfunction by an oxidative stress mechanism in both groups. The magnitude is greater in NIDDM. Fasting HDL-C appears to contribute to the protection of the endothelium against this phenomenon. Hence, exaggerated PPL associated with reduced HDL-C may be important in the pathogenesis of vascular disease, particularly in NIDDM

 

2.20           Rapid identification of LDL subclass phenotypes by iodixanol gradient centrifugation

Davis, I.G. and Griffin, B.A.

Atherosclerosis, 159, 247-252 (2001) 

A rapid and simple method was developed to identify LDL subclass phenotypes for the classification of an atherogenic lipoprotein phenotype. Plasma (3 ml adjusted to 12% iodixanol) was pre-stained (Coomassie Blue R250), under-layered beneath 9% iodixanol and subject to ultracentrifugation (3 h, 65,000 rpm 16°C, (341,000 g)) in a near vertical rotor (Beckman NVT65). A digital photograph of separated LDL bands was downloaded onto a PC and analysed using Total Lab ID gel-scan software (Pharmacia, UK). LDL subclass phenotypes were characterised by Rf values, density intervals and by cross-reference to LDL subclass profiles obtained by our established salt density gradient technique. LDL profiles corresponded to a predominance of light LDL-I, LDL-II or small dense LDL-III. This new method provides a more rapid separation of LDL subclasses than existing salt gradients, with multiple runs and larger rotors (Beckman NVT 65.2 or VTi 65.2) increasing throughput to 48 samples in 24 h.

 

2.21           Direct evidence for a two-step assembly of ApoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes

Cartwright, I.J. and Higgins, J.A.

1. Biol. Chem, 276(51), 48048-48057(2001)

 

The aim of this study was to investigate the types and characteristics of chylomicrons precursor in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low far diet, newly synthesized and Immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.

 

2.22           Superior role of apolipoprotein B48 over apolipoprotein B100 in chylomicron assembly and fat absorption: an investigation of apobec-1 knock-out and wild-type mice.

Kendrick, J.S., Chan, L., and Higgins, J.A.

Biochem. J., 356, 821-827 (2001)

 

Editing of apolipoprotein (apo)-B100 mRNA to yield apo-B48 is a specific and developmentally regulated step in enterocytes of mammals. However, the functional significance of this step is not known. Since mice containing only apo-B100 have not been documented to exhibit any difference in intestinal fat absorption from wild-type mice, the evolutionary advantage of apoB mRNA editing has been questioned. In the present study, we have compared fat absorption and chylomicron assembly in apobec-1 knock-out (KO) or wild-type (WT) mice subjected to different dietary manipulations: low-fat chow, a fat-enriched “Western” diet and overnight fasting. Experiments in vivo and in vitro revealed differences in the ability of KO and WT enterocytes to assemble and secrete chylomicrons under different dietary conditions. After overnight fasting, chylomicrons secretion is reduced considerably in KO compared with WT enterocytes. This is not due to reduced synthesis of apo-B or triacylglycerol (TAG), but appears to be a result of impaired assembly of chylomicrons, so that triacylglycerol accumulates in the enterocytes. After feeding with fat, secretion of chylomicrons enriched in pre-existing TAG is stimulated in KO compared with WT mice. IN the present study, we have documented for the first time that apo-B100 is considerably less efficient that apo-B48 in exerting its role in the early stage of chylomicron assembly, which is rate-limiting in the fat-fed state. Apo-B mRNA editing may result in more efficient fat absorption, specifically under conditions of food shortage or low-fat content, and thus provide an evolutionary advantage.

 

2.23           Vesicle permeabilization by protofibrillar a-synuclein: Implications for the pathogenesis and treatment of Parkinson’s disease

Volles, M.J. et al

Biochemistry, 40, 7812-7819 (2001)

 

Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.

 

2.24           A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass

Sawle, A., Higgins, M.K., Olivant, M.P., and Higgins, J.A.

1. Lipid Res., 43, 335-343 (2002)

 

Determination of the circulating levels of plasma lipoprotein HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoproteins structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electrophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradient were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL, cholesterol, and TG, and identification of LDL subclass pattern.

 

2.25           Persistent and transient replication of full-length hepatitis C virus genomes in cell culture

Pietschmann, T. et al

1. Virol., 76, 4008-4021 (2002)

 

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the en­velope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgl compart­ments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were de­tectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are Important for virus particle assembly and/or release.

 

2.26           Protein interactions with myocilin

Wentz-Hunter, K., Ueda, J. and Yue, B.Y.J.T.

Invest. Ophtalmol. Vis. Sci., 43(1), 176-182 (2002)

PURPOSE. To identify factors that interact in vivo with myocilin,a glaucoma gene product.

METHODS. The yeast two-hybrid system with myocilin as the baitand a human skeletal muscle cDNA library as the prey was usedto identify potential factors that interact with myocilin. Interactionswere also examined in bovine trabecular meshwork (TM) cellsthrough a mammalian two-hybrid system. Biochemical coimmunoprecipitationfrom both human TM cell lysate and in vitro translated proteinswas also used to confirm results obtained from yeast analysis.

RESULTS. Twenty positive clones isolated through yeast two-hybridscreening were deemed potential myocilin partners. Sequenceanalysis determined that two of them encoded for myocilin fromamino acids 64 to 268. Myocilin was also found to interact witha component of the myosin motor protein, myosin regulatory lightchain (RLC). The myocilin–myocilin and myocilin–RLCinteractions revealed by the yeast system were further confirmedand demonstrated in cultured TM cells, by means of a mammaliantwo-hybrid system, and through biochemical coimmunoprecipitation,subcellular fractionation, immunofluorescence, and immunogolddouble labeling.

CONCLUSIONS. These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin–RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.

 

2.27           Evidence for an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial dysfunction and oxidative stress generation

Ceriello, A.C. et al

Circulation, 106, 1211-1218 (2002)

 

Background— Postprandial hypertriglyceridemia and hyperglycemiaare considered risk factors for cardiovascular disease. Evidencesuggests that postprandial hypertriglyceridemia and hyperglycemiainduce endothelial dysfunction through oxidative stress; however,the distinct role of these two factors is a matter of debate.

Methods and Results— Thirty type 2 diabetic patients and20 normal subjects ate 3 different meals: a high-fat meal; 75g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia,nitrotyrosine, and endothelial function were assayed duringthe tests. Subsequently, diabetics took 40 mg/d simvastatinor placebo for 12 weeks. The 3 tests were performed again atbaseline, between 3 to 6 days after the start, and at the endof each study. High-fat load and glucose alone produced a decreaseof endothelial function and an increase of nitrotyrosine innormal and diabetic subjects. These effects were more pronouncedwhen high fat and glucose were combined. Short-term simvastatintreatment had no effect on lipid parameters but reduced theeffect on endothelial function and nitrotyrosine observed duringeach different test. Long-term simvastatin treatment was accompaniedby a lower increase in postprandial triglycerides, which wasfollowed by smaller variations of endothelial function and nitrotyrosineduring the tests.

Conclusions— This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug.

 

2.28           Characterization of HCV RNA particles from the serum of a patient with common variable immunodeficiency on isotonic iodixanol (OptiPrep) gradients. Association with apolipoprotein-B100

Nielsen, S. et al

1. Hepatol., 36, Suppl. 1, 87 (2002)

 

Objective: To investigate the association of HCV RNA and lipoprotein in the serum of an antibody negative patient with common variable immunodeficiency.

Methods: The serum collected six weeks post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection was fractionated by sequential density centrifugation on sodium bromide and by isopycnic centrifugation on isotonic iodixanol (Optiprep).

Results: Whilst sequential density centrifugation yielded HCV RNA fractions of high (> 1.2 g/ml), intermediate (1.063-1.21 g/ml) and low (< 1.063 g/ml) density of approximately equal titre, on iodixanol  gradients all the RNA was recovered at the top of the gradient with a density below 1.13 g/ml. Following precipitation with anti-apoB100 or manganese chloride and heparin the majority of the HCV RNA was removed from the serum leaving only a minor peak with a density of 1.13 g/ml on iodixanol gradients. Treatment of sera with sodium desoxycholate released further particles with a density of 1.13 g/ml in iodixanol gradients, suggesting that this is the density of the virus particle stripped of serum lipoproteins. Treatment with 0.18% NP40 produced a single peak of HCV RNA in a fraction with a density of 1.23 g/ml and a sedimentation coefficient of 150S in iodixanol gradients, taken to be naked viral cores.

Conclusions: Fractionation of HCV RNA on iodixanol in isotonic conditions indicates that essentially all RNA is present in lower density fractions and is associated with low density or very low density lipoproteins.

 

2.29           Characterization of the structural proteins of HCV isolated from human liver

Nielsen, S. et al

1. Hepatol., 36, Suppl. 1, 87 (2002)

 

Objective: To determine the molecular weights of the structural protein of HCV recovered from infected human liver.

Methods: Macerates of a six week post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection were shown to contain 9 LogIU of HCV RNA/g. Macerates were analyzed by isopycnic centrifugation on iodixanol density gradients. HCV RNA was precipitated from crude macerates with manganese chloride and heparin and the solubilised precipitate was analysed by SDS-PAGE and western blotting with monoclonal antibodies to the viral structural proteins.

Results: Following fractionation of the liver macerate on iodixanol density gradients all the HCV RNA was recovered in fractions of density of 1.13 g/ml and below suggesting that the RNA is associated with host lipoprotein. In line with this, manganese/heparin treatment precipitated essentially all of the HCV RNA. SDS-polyacrylamide gel electrophoresis of the proteins present in manganese/heparin precipitates and western blotting revealed a single band of core protein of molecular weight 21 kDa and an E1 band of 31 kDa which migrated in approximately the same position in the gel as the corresponding proteins transiently expressed from a recombinant vaccinia virus system. A single E2 band, however, migrated with a molecular weight of 62 kDa some 8 kDa smaller than the equivalent band expressed from recombinant vaccinia virus.

Conclusions: The molecular weights of the HCV structural proteins recovered from beta-lipoprotein associated virions suggest that processing of the virus polyprotein in the liver may differ from that recombinant vaccinia virus expression system.

 

2.30           Characterization of cytoplasmic a-synuclein aggregates

Lee, H-J. and Lee, S-J.

1. Biol. Chem., 277, 48976-48983 (2002)

 

The a-synuclein fibrillation process has been associated with the pathogenesis of several neuro-degenerative diseases. Here, we have characterized the cytoplasmic a-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of a-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloidlike a-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole

reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the

protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves nonfibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.

 

2.31           Properties of the chaperonin comples from the halophilic archaeon Haloferax volcanii

Large, A.T., Kovacs, E. and Lund, P.A.

FEBS Lett., 532, 309-312 (2002)

 

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75°C. Activity requires high levels of

potassium ions and was reduced in the presence of an increasing concentration of sodium ions.

 

2.32           Methods for measuring lipid metabolism in vivo

Patterson, B. W.

Curr., Opin., Clin., Nutr., Metab. Care, 5, 475-479 (2002)

 

Purpose of review

This review discusses diverse methods that have been used in several recent papers for the qualitative and quantitative analysis of lipids, studies of lipid oxidation, lipoprotein fractionation, and studies of lipid metabolism and metabolic kinetics using tracers. Papers for this review were selected on the basis of their

timeliness, novelty, and/or their potential impact on diverse fields of lipid metabolism.

Recent findings

Many methods used for studies of lipid metabolism employ advanced chromatographic and mass spectrometric techniques to characterize lipids. In particular, the use of stable isotopically labeled tracers has become increasingly important to study metabolic kinetics.

Summary

Such developments in methodology will continue to advance studies of lipid metabolism in many areas of clinical interest, including heart disease, obesity, and diabetes.

 

2.33           Functional reconstitution of purified metabotrophic glutamate receptor expressed in the fly eye

Eroglu, C., Croner, P., Panneels, V., Beaufils, P. and Sinning, I.

EMBO Reports, 3(5), 491-496 (2002)

 

G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.

 

2.34           Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Hu, K. et al

Nature, 415, 646-650 (2002)

 

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

 

2.35           Massive and Selective Delivery of Lipid-Coated Cationic Lipoplexes of Oligonucleotides Targeted in Vivo to Hepatic Endothelial Cells

Bartsch, M., Weeke-Klimp, A.H., Meijer, D.K.F., Scherphof, G.L. and Kamps, J.A.A.M.

Pharmaceutical Res.,19(5), 676-680 (2002)

 

Purpose. Previously we reported on massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver sinusoidal endothelial cells (EC) in vivo. In the present work we applied this principle for the in vivo delivery of antisense oligonucleotides (ODN) to these cells.

Methods. Anti ICAM-1 ODN was complexed with the cationic lipid DOTAP and the complex was coated by an excess of neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was coupled to the coated cationic lipoplexes (CCLs). Plasma disappearance, organ and intrahepatic distribution of Aco-HSA modified CCLs were determined in rats, using [³H]-cholesteryl oleyl ether and ³²P-labeled ODN as markers.

Results. The Aco-HSA coupled CCLs were <160 nm="" in="" size,="" contained="" 1.03="" ±="" 0.35="" nmol="" odn="" and="" 54="" ±="" 18=""> g Aco-HSA per mol total lipid. These CCLs were rapidly eliminated from plasma, about 60% the injected dose of ³H- or ³²P-label being recovered in the liver after 30 min. Within the liver, the EC accounted for two thirds of total liver uptake. Control non-targeted CCLs were eliminated very slowly: after 30 min still >90% of the particles was in the blood.

Conclusions. Our results demonstrate efficient targeting of antisense ODN to EC in vivo, employing plasma-stable coated cationic lipoplexes, surface modified with negatively charged albumin. 40% of the injected ODN was delivered to the target cells within 30 min.

 

2.36           Antioxidant properties of aged garlic extract: an in vitro study incorporating human low density lipoprotein

Dillon, S.A., Burmi, R.S., Lowe, G.M., Billington, D. and Rahman, K.

Life Sciences, 72, 1538-1594 (2003)

 

Oxidation of low-density lipoprotein (LDL) has been recognized as playing an important role in the development and progression of atherosclerotic heart disease. Human LDL was isolated and challenged with a range of oxidants either in the presence or absence of AGE or its diethyl ether extract. Oxidative modification of the LDL fraction using CuSO₄, 5-lipoxygenase and xanthine/xanthine oxidase was monitored by both the appearance of thiobarbituric-acid substances (TBA-RS) and an increase in electrophoretic mobility.This study indicates that AGE is an effective antioxidant as it scavenged superoxide ions and reduced lipid peroxide formation in cell free assays. Superoxide production was completely inhibited in the presence of a 10% (v/v) aqueous preparation of AGE and reduced by 34% in the presence of a 10% (v/v) diethyl ether extract of AGE. The presence of 10% (v/v) diethyl ether extract of AGE significantly reduced Cu²⁺ and 15-lipoxygenase-mediated lipid peroxidation of isolated LDL by 81% and 37%, respectively. In addition, it was found that AGE also had the capacity to chelate copper ions. In contrast, the diethyl ether extract of AGE displayed no copper binding capacity, but demonstrated distinct antioxidant properties. These results support the view that AGE inhibits the in vitro oxidation of isolated LDL by scavenging superoxide and inhibiting the formation of lipid peroxides. AGE was also shown to reduce LDL oxidation by the chelation of Cu²⁺. Thus, AGE may have a role to play in preventing the development and progression of atherosclerotic disease.

 

2.37           Separation of bovine plasma lipoproteins by a rapid ultracentrifugation method

Gardner, R.S., Ogden, N.H., Cripps, P.J. and Billington, D.

1. Comp. Path., 128, 15-23 (2003)

 

The recently described method of centrifugation with iodixanol for the rapid separation of human plasma lipoproteins was adapted to separate bovine plasma lipoproteins. Density gradients were generated by mixing plasma with iodixanol 12% (w/v), followed by centrifugation at 350000g and 16 degrees C for 3h 10min in a vertical rotor. Gradients were unloaded dense-end first into 10 fractions. Human very low density lipoprotein (VLDL; density <1.011g/ml), low density lipoprotein (LDL; density = 1.016-1.039 g/ml) and high density lipoprotein (HDL; density = 1.039-1.090 g/ml) were resolved well at densities considerably lower than those traditionally reported in salt gradients. In gradients generated from 12% iodixanol, bovine LDL and HDL exhibited even lower densities (1.016-1.028 and 1.016-1.048g/ml, respectively) with all lipoproteins occurring at the lower density region of the gradient. In contrast, density gradients generated from layers of equal volumes of 6% and 12% iodixanol readily separated bovine HDL from VLDL, whilst LDL still overlapped with HDL. The latter accounts for >80% of all bovine lipoproteins and exists as two populations, namely light and heavy HDL. Gradients generated from two layers of iodixanol recovered bovine HDL in five fractions. The hypercholesterolaemia associated with lactation resulted in a modest shift in the profile of HDL cholesterol towards lipoprotein particles of lower density (light HDL). Significant between-farm differences were also detected in the density profiles of bovine plasma cholesterol. This new method is suitable for use in research and diagnosis in relation to lipoprotein metabolism disorders in cows.

 

2.38           Characterization of the genome and structural proteins of b-lipoprotein associated HCV extracted from infected human liver

Nielsen, S., Bassendine, M.F., Burt, A. And Toms, G.L.

GUT, Abstract from the British association for the study of liver meeting 2002, abstract 94 (2003)

 

Serum and liver macerates from a patient with common variable immunodeficiency and chronic HCV infection six weeks post-transplant were analysed by isopycnic centrifugation on isotonic iodixanol (optiprep) density gradients. All of the HCV RNA fractionated at the top of the gradient, in fractions of density of < 1.13 g/ml and was precipitable with manganese chloride and heparin (Mn/Hep) indicating that it is all associated with host beta-lipoprotein. Treatment with desoxycholate released putative hepatitis C virions with a density of 1.13 g/ml and treatment with 0.18% NP40 released putative virus cores with a density of 1.21 g/ml and a sedimentation coefficient of 150S. Northern blotting of Mn/Hep precipitates revealed a single band of HCV RNA of 9.4 kb corresponding in size to the full HCV genome. SDS-polyacrylamide gel electrophoresis and western blotting with monoclonal antibodies revealed a single 20 kDa band of core protein and a single 31 kDa E1 band reduced to 20 kDa after deglycosylation with endoglycosidase F. Both core and E1 bands co-migrated with corresponding bands derived from a recombinant vaccinia virus system expressing the HCV structural protein genes. Anti-E2 Mabs blotted a single 62 kDa band from Mn/Hep precipitates, approximately 7 kDa smaller than the anti-E2 staining band in the recombinant vaccinia virus system. Following deglycosylation, E2 glycoprotein ran with an apparent molecular weight of 36 kDa, co-migrating with E2, which forms a minor band in the deglycosylated vaccinia virus recombinant. No band equivalent to the major 40 kDa E2/P7 band in the deglycosylated vaccinia virus system was observed in the Mn/Hep precipitates, indicating that E2/P7 is not a structural protein. Under reducing conditions both E1 and E2 ran as monomers, suggesting that the two glycoproteins are not disulphide linked in the virion.

 

2.39           A novel role for CD36 in VLDL-enhanced platelet activation

Englyst, N.A., Taube, J.M., Aitman, T.J., Baglin, T.P. and Byrne C.D.

Diabetes, 52, 1248-1255 (2003)

 

Type 2 diabetes is characterized by increased plasma triglyceride levels and a fourfold increase in ischemic heart disease, but the mechanism is unclear. CD36 is a receptor/transporter that binds fatty acids of lipoproteins. CD36 deficiency has been linked with insulin resistance. There is strong evidence of in vivo interaction between platelets and atherogenic lipoproteins suggesting that atherogenic triglyceride-rich lipoproteins, such as VLDL, that are increased in diabetic dyslipidemia are important in this process. This study demonstrates that VLDL binds to the platelet receptor CD36, enhances platelet thromboxane A2 production, and causes increased collagen-mediated platelet aggregation. VLDL enhanced collagen-induced platelet aggregation by 1) shortening the time taken for aggregation to begin (lag time) to 70% of control (P = 0.001); 2) increasing maximum aggregation to 170% of control (P = 0.008); and 3) increasing thromboxane production to 3,318% of control (P = 0.004), where control represents platelets stimulated with collagen (100%). A monoclonal antibody against CD36 attenuated VLDL-enhanced collagen-induced platelet aggregation by 1) inhibiting binding of VLDL to platelets by 75% (P = 0.041); 2) lengthening lag time to 190% (P < 0.001); and 3) decreasing thromboxane production to 8% of control (P < 0.001). In support of this finding, platelets from Cd36-deficient rats showed no increase in aggregation, thromboxane production, and VLDL binding in contrast to platelets from rats expressing CD36. These data suggest that platelet Cd36 has a key role in VLDL-induced collagen-mediated platelet aggregation, possibly contributing to atherothrombosis associated with increased VLDL levels.

 

2.40           Rapid separation of LDL subclasses by iodixanol gradient ultracentrifugation

Davies, I.G., Graham, J.M. and Griffin, B.A.

Clin. Chem., 49(11), 1865-1872 (2003)

 

Background: A predominance of small, dense LDL (sdLDL) confersin excess of a threefold increase in coronary heart disease(CHD) risk. The conventional method for the detection of sdLDL,salt density gradient ultracentrifugation (DGUC) has been supersededby more rapid techniques. This report presents novel methodologyfor the separation of sdLDL by a combination of iodixanol densitygradient centrifugation and digital photography.

Methods: LDL subclasses were separated in 3 h from prestainedplasma on a self-forming density gradient of iodixanol. LDLsubclass profiles were generated by digital photography andgel-scan software. Plasma samples from 106 normo- and dyslipidemicindividuals were used to optimize the gradient for the resolutionof LDL heterogeneity. A subgroup of 47 LDL profiles were thencompared with LDL subclasses separated by salt DGUC.

Results: The peak density of the predominant LDL band correlated significantly with the relative abundance (as a percentage) of sdLDL as resolved by salt DGUC (P <0.001). As shown previously, LDL isolated at a lighter density in iodixanol compared with salt gradients. A predominance of sdLDL corresponded to a peak density on iodixanol of 1.028 kg/L. This density and the area under the LDL profile lying above this density were sensitive and specific markers for the prediction of a predominance of sdLDL (P <0.001) and showed predictable associations with plasma triglycerides (r = 0.59; P <0.001) and HDL (r = -0.4; P <0.001).

Conclusions: This simple method for the detection of sdLDL candifferentiate a predominance of sdLDL, is highly reproducible,and can be used preparatively to isolate sdLDL.

 

2.41           Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archeon Arcidianus ambivalens

Moll, R.G.

Biochem. J., 374, 247-254 (2003)

 

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP–RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic super-natants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic super-natants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

 

2.42           The effect of CTAB concentration in cationic PLG microparticles on DNA adsorption and in vivo performance

Singh, M. et al

Pharmaceut, Res., 20(2), 247-251 (2003)

 

Purpose. Cationic PLG microparticles with adsorbed DNA have previously been shown to efficiently target antigen presenting cells in vivo for generating higher immune responses in comparison to naked DNA. In this study we tried to establish the role of surfactant (CTAB) concentration on the physical behavior of these formulations.

Methods. Cationic PLG microparticle formulations with adsorbed DNA were prepared using a solvent evaporation technique. Formulations with varying CTAB concentrations and a fixed DNA load were prepared. The loading efficiency and 24 h DNA release was evaluated for each formulation. Select formulations were tested in vivo.

Results. Higher CTAB concentration correlated with higher DNA binding efficiency on the microparticles and lower in vitro release rates. Surprisingly though, the in vivo performance of formulations with varying CTAB concentration was comparable to one another.

Conclusions. Cationic PLG microparticles with adsorbed DNA, as described here, offer a robust way of enhancing in vivo responses to plasmid DNA.

 

2.43           Trans Unsaturated Fatty Acids Are Less Oxidizable than Cis Unsaturated Fatty Acids and Protect Endogenous Lipids from Oxidation in Lipoproteins and Lipid Bilayers

Sargis, R.M. and Subbaiah, P.V.

Biochemistry, 42, 11533-11543 (2003)

 

Epidemiological data suggest that dietary trans unsaturated fatty acids increase the risk of heart disease; however, the underlying mechanisms are unclear. In this study, we investigated one possible mechanism, namely, their effect on LDL oxidation. Supplementation of LDL with 10% 16:1 trans-cholesteryl ester (CE) inhibited the oxidation compared to that with 16:1 cis-CE. Total replacement of core lipids with 18:2 trans,trans-CE decreased the rate of LDL oxidation by 19% compared to replacement with 18:2 cis,cis-CE. When the surface phosphoglycerides were replaced with either 16:0-18:2 cis,cis-phosphatidylcholine (PC) or 16:0-18:2 trans,trans-PC, the latter was found to inhibit the rate and increase the lag time of oxidation to a greater extent than the former. To confirm these findings, we studied the oxidation of PC liposomes by assessing the formation of conjugated dienes or the degradation of a fluorescently labeled PC. By both methods, the 16:0-18:2 trans,trans-PC exhibited greater resistance to oxidation than the 16:0-18:2 cis,cis-PC. Eliminating the fluidity differences did not completely eliminate the differences in oxidation rates, suggesting that the trans double bond is inherently resistant to oxidation. The composition of the conjugated hydroperoxy products formed after oxidation differed markedly for the two 18:2 isomers. Supplementation of 16:0-18:2 cis,cis-PC liposomes with 20 mol % di16:1 trans-PC retarded oxidation rates to a greater extent than supplementation with di16:1 cis-PC. These studies show that dietary trans unsaturated fatty acids decrease the rate of lipid peroxidation, an effect that may mitigate the atherogenic effect of these fatty acids.

 

2.44           Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation

Nakano, E., Williamson, M.P., Williams, N.H. and Powers, H.J.

Biochim. Biophys. Acta, 1688, 33-42 (2004)

 

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 M) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.

 

2.45           Effect of postprandial hypertriglyceridemia and hyperglycemia on circulating adhesion molecules and oxidative stress generation and the possible role of  simvastatin treatment

Ceriello, A. et al

Diabetes, 53, 701-710 (2004)

 

Adhesion molecules, particularly intracellular adhesion molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin,have been associated with cardiovascular disease. Elevated levelsof these molecules have been reported in diabetic patients.Postprandial hypertriglyceridemia and hyperglycemia are consideredrisk factors for cardiovascular disease, and evidence suggeststhat postprandial hypertriglyceridemia and hyperglycemia mayinduce an increase in circulating adhesion molecules. However,the distinct role of these two factors is a matter of debate.Thirty type 2 diabetic patients and 20 normal subjects ate threedifferent meals: a high-fat meal, 75 g of glucose alone, anda high-fat meal plus glucose. Glycemia, triglyceridemia, plasmanitrotyrosine, ICAM-1, VCAM-1, and E-selectin were assayed duringthe tests. Subsequently, diabetic subjects took simvastatin40 mg/day or placebo for 12 weeks. The three tests were performedagain at baseline, between 3 and 6 days after starting the study,and at the end of each study. High-fat load and glucose aloneproduced an increase of nitrotyrosine, ICAM-1, VCAM-1, and E-selectinplasma levels in normal and diabetic subjects. These effectswere more pronounced when high fat and glucose were combined.Short-term simvastatin treatment had no effect on lipid parameters,but reduced the effect on adhesion molecules and nitrotyrosine,which was observed during every different test. Long-term simvastatintreatment was accompanied by a lower increase in postprandialtriglycerides, which was followed by smaller variations in ICAM-1,VCAM-1, E-selectin, and nitrotyrosine during the tests. Thisstudy shows an independent and cumulative effect of postprandialhypertriglyceridemia and hyperglycemia on ICAM-1, VCAM-1, andE-selectin plasma levels, suggesting oxidative stress as a commonmediator of such effects. Simvastatin shows a beneficial effecton oxidative stress and the plasma levels of adhesion molecules,which may be ascribed to a direct effect in addition to thelipid-lowering action of the drug.

 

2.46           Effects of Rosiglitazone on endothelial function in men with coronary artery disease without diabetes mellitus

Sidhu, J.S., Cowan, D. and Kaski, J.C.

Am. J. Cardiol., 94, 151-156 (2004)

 

Recent data have shown that peroxisome proliferator-activated receptor- agonists may exert protective effects on the vascular endothelium by amelioration of insulin resistance and through direct anti-inflammatory effects. In this study we assessed the effect of rosiglitazone on biochemical and biophysical indexes of endothelial function in male, nondiabetic patients with coronary artery disease. Consecutive male subjects (n = 71) with clinically stable, angiographically documented coronary artery disease and without diabetes mellitus were investigated. Patients were randomized in a double-blind manner to placebo or rosiglitazone for a total of 24 weeks. Flow-mediated dilation (FMD) of the brachial artery, C-reactive protein, von Willebrand factor, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels, and parameters of glucose and lipid metabolism were measured at baseline and after 12 and 24 weeks of treatment. Rosiglitazone treatment significantly reduced C-reactive protein (median 0.56 mg/L [interquartile range 0.33 to 1.02] to 0.33 mg/L [interquartile range 0.26 to 0.40], p <0.01), von Willebrand factor (139 ± 47 to 132 ± 44 IU/dl, P = 0.02), insulin resistance index (p = 0.05), and mean low-density lipoprotein (LDL) density (p <0.001) compared with placebo. However, no significant differences were seen between the rosiglitazone and placebo groups with regard to brachial artery FMD, intercellular adhesion molecule-1, or vascular cell adhesion molecule-1 levels. Rosiglitazone treatment significantly increased LDL (2.62 ± 0.72 to 2.95 ± 0.84 mmol/L, P = 0.03) and triglyceride (1.23 ± 0.63 to 1.56 ± 0.98 mmol/L, P = 0.04) levels. Thus, rosiglitazone reduced markers of inflammation and endothelial activation, but this did not translate into an improvement in FMD. Increased LDL and triglyceride levels may have played a role.

 

2.47           Plasma appearance of unesterified astaxanthin geometrical E/Z and optical R/S isomers in men given single doses of a micture of optical 3 and 3’R/S isomers of astaxanthin fatty acyl diesters

Coral-Hinostroza, G.N., Ytrestøyl, T., Ruyter, B. and Bjerkeng, B.

Comp. Biochem. Biophys.Part C, 139, 99-110 (2004)

 

Appearance, pharmacokinetics and distribution of astaxanthin all-E-, 9Z- and 13Z-geometrical and (3R,3′R)-, (3R,3′S)- and (3S,3′S)-optical isomers in plasma fractions were studied in three middle-aged male volunteers (41–50 years) after ingestion of a single meal containing first a 10-mg dose equivalent of astaxanthin from astaxanthin diesters, followed by a dose of 100 mg astaxanthin equivalents after 4 weeks. Direct resolution of geometrical isomers and optical isomers of astaxanthin dicamphanates by HPLC after saponification showed that the astaxanthin consisted of 95.2% all-E-, 1.2% 9Z- and 3.6% 13Z-astaxanthin, of (3R,3′R)-, (3R,3′S; meso)- and (3S,3′S)-astaxanthin in a 31:49:20 ratio. The plasma astaxanthin concentration–time curves were measured during 76 h. Astaxanthin esters were not detected in plasma. Maximum levels of astaxanthin (C_max=0.28±0.1 mg/l) were reached 11.5 h after administration and the plasma astaxanthin elimination half-life was 52±40 h. The C_max at the low dose was 0.08 mg/l and showed that, the dose response was non-linear. The (3R,3′R)-astaxanthin optical isomer accumulated selectively in plasma compared to the (3R,3′S)- and (3S,3′S)-isomers, and comprised 54% of total astaxanthin in the blood and only 31% of total astaxanthin in the administered dose. The astaxanthin Z-isomers were absorbed selectively into plasma and comprised 32% of total astaxanthin 6–7.5 h postprandially. The proportion of all-E-astaxanthin was significantly higher in the very low density lipoproteins and chylomicrons (VLDL/CM) plasma lipoprotein fraction than in the high density lipoproteins (HDL) and low denisty lipoproteins (LDL) fractions (P<0.05). The results indicate that a selective process increase the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin before uptake in blood and that the astaxanthin esters are hydrolyzed selectively during absorption.

 

2.48           Prevention of Alzheimer’s disease-associated Ab aggregation by rationally designed nonpeptidic b-sheet logands

Rzepecki, P. et al

1. Biol. Chem., 279(46), 47497-47505 (2004)

 

A new concept is introduced for the rational design of -sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a -sheet efficiently block the solvent-exposed -sheet portions in A -(1–40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of A aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a -sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10^–5 M. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of A -(1–40) in the absence and in the presence of the ligands. From the comparable decrease in A monomer concentration, we conclude that these -sheet ligands do not prevent the initial oligomerization of monomeric A but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble A aggregates formed in the presence of both inhibitors from 3- to 15-mers.

 

2.49           A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes

Hu, K., Rickman, C., Carroll, J. and Davletov, B.

Biochem. J., 377, 781-795 (2004)

 

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.

 

2.50           Hyperlipidemic subjects have reduced uptake of newly absorbed vitamin E into their plasma lipoproteins, erythrocytes, platelets, and lymphocytes, as studied by deuterium-labeled a-tocopherol biokinetics

Hall, W.L., Jeanes, Y.M. and Lodge, J.K.

1. Nutr., 135, 58-63 (2005)

 

Vitamin E homeostasis in hyperlipidemia is poorly understood. The biokinetics of deuterated -tocopherol ( -T) in blood components was investigated in normolipidemic (N; total cholesterol < 5.5 mmol/L and triglycerides < 1.5 mmol/L, n = 9), hypercholesterolemic (HC; total cholesterol > 6.5 mmol/L and triglycerides < 1.5 mmol/L, n = 10), and combined hypercholesterolemic and hypertriglyceridemic (HCT; total cholesterol > 6.5 mmol/L and triglycerides > 2.5 mmol/L, n = 6) subjects. Subjects ingested 150 mg hexadeuterated RRR- -tocopheryl acetate, and blood was collected up to 48 h after ingestion. Labeled -T was measured in plasma, lipoproteins, erythrocytes, platelets, and lymphocytes by liquid chromatography/mass spectroscopy. In plasma, HC had an earlier time of maximum concentration (6 h) compared with N and HCT (12 h) (P < 0.05). HCT had a lower uptake of labeled -T (P < 0.005) and a longer half-life (P < 0.05). In chylomicrons, the maximum labeled -T concentration was higher in HC compared with N and HCT (P < 0.00005); however, HCT had a lower uptake of labeled -T in LDL. In all groups, the lowest density LDL subfraction contained more labeled -T than denser subfractions (P < 0.05). In platelets, lymphocytes, and erythrocytes, the areas under the labeled -T concentration vs. time curves were in the order N > HC > HCT. In lymphocytes, differences in labeled -T were found at 6 and 48 h (P < 0.05). These data demonstrate that there are differences in the uptake of newly absorbed -T into blood components in hyperlipidemia. Because these blood components are functionally affected by vitamin E, reduced uptake of -T may be relevant to the pathogenesis of atherosclerosis.

  

2.51           Cell surface heparan sulfate proteoglycans contribute to intracellular lipid accumulation in adipocytes

Wilsie, L.C., Chanchani, S., Navaratna, D. and Orlando, R.A.

Lipids in Health and Disease, 4(2), 1-15 (2005)

 

Background

Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).

Results

We found that expression of these three lipoprotein receptors increased 5-fold, 2-fold, and 2.5-fold, respectively, during adipocyte differentiation. The major proteoglycans expressed by mature adipocytes are of high molecular weight (>500 kD) and contain both heparan and chondroitin sulfate moieties. Using ligand binding antagonists, we observed that HSPG, rather than VLDL-R or LRP, play a primary role in the uptake of DiI-lableled apoE-VLDL by mature adipocytes. In addition, inhibitors of HSPG maturation resulted in a significant reduction (>85%) in intracellular lipid accumulation.

Conclusions

These results suggest that cell surface HSPG is required for fatty acid transport across the plasma membrane of adipocytes.

 

2.52           Optimized targeting of polyethylene glycol-stabilized anti-intercellular adhesion molecule 1 oligonucleotide/lipid particles to liver sinusoidal endothelial cells

Bartsch, M. et al

Mol. Pharmacol., 67(3), 883-890 (2005)

 

We prepared polyethylene glycol (PEG)-stabilized antisense oligonucleotide (ODN)/lipid particles from a lipid mixture including the positively charged amphiphile 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and anti-intercellular adhesion molecule 1 (ICAM-1) antisense ODN by an extrusion method in the presence of 40% ethanol. These particles were targeted to scavenger receptors on liver endothelial cells by means of covalently coupled polyanionized albumin. Two types of such targeted particles were prepared, one with the albumin coupled to a maleimide group attached to the particle's lipid bilayer and the other with the protein coupled to a maleimide group attached at the distal end of added bilayer-anchored PEG chains. Upon intravenous injection, the ODN particles with bilayer-coupled albumin were cleared from the blood circulation at the same low rate as untargeted particles (<5% in 30 min). By contrast, the distal-end coupled particles were very rapidly cleared from the blood and preferentially taken up by the endothelial cells of the hepatic sinusoid (55% of injected dose after 30 min). Despite this substantial endothelial targeting, no consistent inhibition of ICAM-1 expression could be demonstrated in this cell type, either in vivo or in vitro. However, in J774 cells that also express scavenger receptors and ICAM-1, significant down-regulation of ICAM-1 mRNA was achieved with distal-end targeted lipid particles, as determined with real-time RT-PCR. It is concluded that massive delivery of ODN to cell types that express scavenger receptors can be achieved if lipid particles are provided with negatively charged albumin distally attached to bilayer anchored PEG chains.

 

2.53           Downstream effects on human low density lipoprotein of homoocysteine exported from endothelial cells in an in vitro system

Nakano, E. et al

1. Lipid Res., 46, 484-493 (2005)

 

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposedto HUVECs exporting Hcy undergo time-related lipid oxidation,a process inhibited by the thiol trap dithionitrobenzoate. Thisis likely to be related to the generation of hydroxyl radicals,which we show are associated with Hcy export. Although Hcy isthe major oxidant, cysteine also contributes, as shown by theeffect of glutamate. Finally, the LDL oxidized in this systemshowed a time-dependent increase in uptake by human macrophages,implying an upregulation of the scavenger receptor.

These results suggest that continuous export of Hcy from endothelialcells contributes to the generation of extracellular hydroxylradicals, with associated oxidative modification of LDL andincorporation into macrophages, a key step in atherosclerosis.Factors that regulate intracellular Hcy metabolism modulatethese effects.

 

2.54           Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer’s disease PET imaging agents on b-amyloid peptide fibrils

Lockhart, A. et al

1. Biol. Chem., 280(9), 7677-7684 (2005)

 

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of -amyloid peptide (A )-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated A -(1–40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the A fibrils. All of the compounds bound with very high affinity (low nM K_d) to a low capacity site (BS3) (1 ligand-binding site per 300 A -(1–40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K_d 100 nM) to either one of two additional binding sites on the A -(1–40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every 35 or every 4 monomers of A -(1–40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.

 

2.55           Distinct signaling particles containing ERK/MEK and B-Raf in PC12 cells

MacCormick, M. et al

Biochem. J., 387, 155-164 (2005)

 

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated ‘signalling particles’ with an estimated size of 60–75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

 

2.56           A Dictyostelium homologue of WASP is required for polarized F-actin assembly during chemotaxis

Myers, S.A., Han, J.W., Lee, Y., Firtel, R.A. and Chung, C.Y.

Mol. Biol. Cell, 16, 2191-2206 (2005)

 

The actin cytoskeleton controls the overall structure of cells and is highly polarized in chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a lesser degree in the cell's posterior. Wiskott-Aldrich syndrome protein (WASP) has emerged as a central player in controlling actin polymerization. We have investigated WASP function and its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of WASP is required for the localization of WASP. We demonstrated that the B domain binds to PI(4,5)P₂ and PI(3,4,5)P₃ with similar affinities. The interaction between the B domain and PI(3,4,5)P₃ plays an important role for the localization of WASP to the leading edge in chemotaxing cells. Our results suggest that the spatial and temporal control of WASP localization and activation is essential for the regulation of directional motility.

 

2.57           Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A

Rashid, D.J., Bononi, J., Tripet, B.P., Hodges, R.S. and Pierce, D.W.

J.Peptide Res., 65, 538-549 (2005)

 

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.

 

2.58           Effect of Atorvastatin and Irbesartan, alone and in combination, on postprandial endothelial dysfunction, oxidative stress, and inflammation in type 2 diabetic patients

Ceriello, A. et al

Circulation, 111, 2518-2524 (2005)

 

Background— Postprandial hypertriglyceridemia and hyperglycemiaare considered risk factors for cardiovascular disease. Evidencesuggests that postprandial hypertriglyceridemia and hyperglycemiainduce endothelial dysfunction and inflammation through oxidativestress. Statins and angiotensin type 1 receptor blockers havebeen shown to reduce oxidative stress and inflammation, improvingendothelial function.

Methods and Results— Twenty type 2 diabetic patients ate3 different test meals: a high-fat meal, 75 g glucose alone,and a high-fat meal plus glucose. Glycemia, triglyceridemia,endothelial function, nitrotyrosine, C-reactive protein, intercellularadhesion molecule-1, and interleukin-6 were assayed during thetests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan300 mg/d, both, or placebo for 1 week. The 3 tests were performedagain between 5 and 7 days after the start of each treatment.High-fat load and glucose alone produced a decrease in endothelialfunction and increases in nitrotyrosine, C-reactive protein,intercellular adhesion molecule-1, and interleukin-6. Theseeffects were more pronounced when high-fat load and glucosewere combined. Short-term atorvastatin and irbesartan treatmentssignificantly counterbalanced these phenomena, and their combinationwas more effective than either therapy alone.

Conclusions— This study confirms an independent and cumulativeeffect of postprandial hypertriglyceridemia and hyperglycemiaon endothelial function and inflammation, suggesting oxidativestress as a common mediator of such an effect. Short-term treatmentwith atorvastatin and irbesartan may counterbalance this phenomenon;the combination of the 2 compounds is most effective.

 

2.59           Distribution of brevetoxin (PbTx-3) in mouse plasma: association with high-density lipoprotein

Woofter, R.T., Spiess, P.C. and Ramsdell. J.S.

Environ. Health Perspect., 113(11), 1491-1496 (2005)

 

We investigated the brevetoxin congener PbTx-3 to determine its distribution among carrier proteins, including albumin and blood lipoproteins. Using a radiolabeled brevetoxin tracer (PbTx-3), we found that 39% of the radiolabel remained associated with components in mouse plasma after > 15 kDa cutoff dialysis. Of this portion, only 6.8% was bound to serum albumin. We also examined the binding of brevetoxin to various lipoprotein fractions. Plasma, either spiked with PbTx-3 or from mice treated for 30 min with PbTx-3, was fractionated into different-sized lipoproteins by iodixanol gradient ultracentrifugation. Each fraction was then characterized and quantified by agarose gel electrophoresis and brevetoxin radioimmunoassay, respectively. In both the in vitro and in vivo experiments, the majority of brevetoxin immunoreactivity was restricted to only those gradient fractions that contained high-density lipoproteins (HDLs). Independent confirmation of brevetoxin binding to HDLs was provided by high molecular weight (100 kDa cutoff) dialysis of [3H]PbTx-3 from lipoprotein fractions as well as a scintillation proximity assay using [3H]PbTx-3 and purified human HDLs. This information on the association of brevetoxins with HDLs provides a new foundation for understanding the process by which the toxin is delivered to and removed from tissues and may permit more effective therapeutic measures to treat intoxication from brevetoxins and the related ciguatoxins.

 

2.60           Phosphorylation-induced autoinhibition regulates the cytoskeletal protein lethal (2) giant larvae

Betschinger, J., Eisenhaber, F. and Knoblich, J.A.

Current Biol., 15(3), 276-282 (2005)

 

During asymmetric cell division, cell fate determinants localize asymmetrically and segregate into one of the two daughter cells. In Drosophila neuroblasts, the asymmetric localization of cell fate determinants to the basal cell cortex requires aPKC. aPKC localizes to the apical cell cortex and phosphorylates the cytoskeletal protein Lethal (2) giant larvae (Lgl). Upon phosphorylation, Lgl dissociates from the cytoskeleton and becomes inactive. Here, we show that phosphorylation regulates Lgl by allowing an autoinhibitory interaction of the N terminus with the C terminus of the protein. We demonstrate that interaction with the cytoskeleton is mediated by a C-terminal domain while the N terminus is not required. Instead, the N terminus can bind to the C terminus and can compete for binding to the cytoskeleton. Interaction between the N- and C-terminal domains requires phosphorylation of Lgl by aPKC. Our results suggest that unphosphorylated, active Lgl exists in an open conformation that interacts with the cytoskeleton while phosphorylation changes the protein to an autoinhibited state.

 

2.61           E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

Perez, T.D., Nelson, W.J., Boxer, S.G. and Kam, L.

Langmuir, 21, 11963-11968 (2005)

 

Cell−cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm²/s and mobile fraction of 30−60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

 

2.62           A discoidal lipoprotein from the coelomic fluid of the polychaete Nereis virens

Schenk, S., Harris, J.R. and Hoeger, U.

Comp. Biochem. Physiol., Part B, 143, 236-243 (2006)

 

A discoidal lipoprotein was isolated from the coelomic fluid of the polychaete, Nereis virens, by density gradient centrifugation. The lipoprotein was present in both sexes and moved as a uniform band in an agarose gel. The average diameter of the lipoprotein particles determined by electron microscopy was 42 nm with a thickness of 10 nm. SDS electrophoresis showed two apoprotein subunits with molecular masses of 247 and 85 kDa, respectively. In lectin blots, both apoproteins were reactive with Concanavalin A indicating the presence of N-glycans. The small subunit was also reactive with peanut lectin, indicating additional O-glycosylation. The total lipid content was 48% and consisted mainly of phospholipids and some diglycerides as judged by thin layer chromatography. The estimated native molecular mass of N. virens lipoprotein ( 675 kDa) lies in the range of vertebrate high-density lipoprotein and insect lipophorins. The size of the apoproteins is similar to those found in insects, while the composition of the lipid fraction is more similar to that of crustacean lipoproteins.

 

2.63           Blood lipid concentrations and lipoprotein patterns in captive and wild American black bears (Ursus americanus)

Frank, N., Elliott, S.B., Allin, S.B. and Ramsay, E.C.

Am. J. Vet. Res., 67(2), 335-341 (2006)

 

Objective-To compare blood lipid concentrations and lipoprotein patterns for captive and wild American black bears (Ursus americanus). Animals-7 captive and 9 wild adult (>/= 4 years old) black bears. Procedure-Blood was collected from 2 groups of captive black bears (groups A and B) and 1 group of wild black bears (group C). Blood triglyceride (TG) and cholesterol concentrations were compared among groups. Plasma lipoproteins were isolated by use of a self-generating gradient of iodixanol, and lipoprotein patterns were compared between groups A and B. Results-Captive bears (mean +/- SD, 187.8 +/- 44.4 kg) weighed significantly more than wild bears (mean, 104.8 +/- 41.4 kg), but mean body weight did not differ between groups A and B. Mean blood TG concentrations for groups B (216.8 +/- 16.0 mg/dL) and C (190.7 +/- 34.0 mg/dL) were significantly higher than that of group A (103.9 +/- 25.3 mg/dL). Mean blood cholesterol concentration was also significantly higher for group B (227.8 +/- 8.2 mg/dL) than for groups A (171.7 +/- 35.5 mg/dL) or C (190.8 +/- 26.8 mg/dL). Mean very-low-density lipoprotein TG and low-density lipoprotein cholesterol concentrations were 2- and 3-fold higher, respectively, for group B, compared with concentrations for group A. Conclusions and Clinical Relevance-Blood lipid concentrations vary significantly among populations of black bears. Plasma lipoprotein patterns of captive bears differed significantly between colonies and may have reflected differences in diet or management practices.

 

2.64           Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated by vitamin C in type 2 diabetes

Anderson, R.A. et al

Diabetic Med., 23, 258-264 (2006)

 

Background  Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C.

Methods  Twenty subjects with T2DM with moderate glycaemic control (mean HbA_1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery.

Results  There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 ± 1.3% to 4 h 0.22 ± 1.1% (P < 0.05) and 8 h 0.7 ± 0.9% (P < 0.05) (mean ± sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 ± 0.9–2.8 ± 0.8 (at 4 h) and 2.9 ± 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 ± 118 (B) to 224.7 ± 72 P < 0.05] and not at 8 h 301.1 ± 118 (B) to 260 ± 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 ± 1 to 2.7 ± 1 at 4 h and 1.95 ± 1.2 at 8 h (P = 0.0002 and 0.33, respectively).

Conclusion  PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.

 

2.65           Soy-isoflavone-enriched foods and markers of lipid and glucose metabolism in postmenopausal women: interactions with genotype and equol production

Hall, W.L. et al

Am. J. Clin. Nutr., 83, 592-600 (2006)

 

Background: The hypocholesterolemic effects of soy foods arewell established, and it has been suggested that isoflavonesare responsible for this effect. However, beneficial effectsof isolated isoflavones on lipid biomarkers of cardiovasculardisease risk have not yet been shown.

Objective: The objective was to investigate the effects of isolated soy isoflavones on metabolic biomarkers of cardiovascular disease risk, including plasma total, HDL, and LDL cholesterol; triacylglycerols; lipoprotein(a); the percentage of small dense LDL; glucose; nonesterified fatty acids; insulin; and the homeostasis model assessment of insulin resistance. Differences with respect to single nucleotide polymorphisms in selected genes [ie, estrogen receptor (XbaI and PvuII), estrogen receptor ß (AluI), and estrogen receptor ß(cx) (Tsp509I), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), cholesteryl ester transfer protein (TaqIB), andleptin receptor (Gln223Arg)] and with respect to equol productionwere investigated.

Design: Healthy postmenopausal women (n = 117) participatedin a randomized, double-blind, placebo-controlled, crossoverdietary intervention trial. Isoflavone-enriched (genistein-to-daidzeinratio of 2:1; 50 mg/d) or placebo cereal bars were consumedfor 8 wk, with a wash-out period of 8 wk before the crossover.

Results: Isoflavones did not have a significant beneficial effect on plasma concentrations of lipids, glucose, or insulin. A significant difference between the responses of HDL cholesterol to isoflavones and to placebo was found with estrogen receptor ß(cx) Tsp509I genotype AA, but not GG or GA.

Conclusions: Isoflavone supplementation, when provided in theform and dose used in this study, had no effect on lipid orother metabolic biomarkers of cardiovascular disease risk inpostmenopausal women but may increase HDL cholesterol in anestrogen receptor ß gene–polymorphic subgroup.

 

2.66           Inducible expression of Tau repeat domain in cell models of tauopathy

Khlistunova, I. et al

1. Biol. Chem., 281(2), 1205-1214 (2006)

 

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau_RD) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation K280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with K280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau_RD is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for -structure. (c) Soluble mutants of tau_RD that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau_RD in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau_RD (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau_RD disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau_RD that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

 

2.67           Effects of dairy products naturally enriched with cis-9, trans-11 conjugated linoleic acid on the blood lipid profile in healthy middle-aged men

Tricon, S. et al

Am. J. Clin. Nutr., 83, 744-753 (2006)

 

Background: Interest in the development of dairy products naturally enriched in conjugated linoleic acid (CLA) exists. However, feeding regimens that enhance the CLA content of milk also increase concentrations of trans-18:1 fatty acids. The implications forhuman health are not yet known.

Objective: This study investigated the effects of consuming dairy products naturally enriched in cis-9,trans-11 CLA (and trans-11 18:1) on the blood lipid profile, the atherogenicityof LDL, and markers of inflammation and insulin resistance inhealthy middle-aged men.

Design: Healthy middle-aged men (n = 32) consumed ultra-heat-treated milk, butter, and cheese that provided 0.151 g/d (control) or 1.421 g/d (modified) cis-9,trans-11 CLA for 6 wk. This was followedby a 7-wk washout and a crossover to the other treatment.

Results: Consumption of dairy products enriched with cis-9,trans-11 CLA and trans-11 18:1 did not significantly affect body weight, inflammatory markers, insulin, glucose, triacylglycerols, or total, LDL, and HDL cholesterol but resulted in a small increase in the ratio of LDL to HDL cholesterol. The modified dairy products changed LDL fatty acid composition but had no significant effect on LDL particle size or the susceptibility of LDL to oxidation. Overall, increased consumption of full-fat dairy products and naturally derived trans fatty acids did not cause significantchanges in cardiovascular disease risk variables, as may beexpected on the basis of current health recommendations.

Conclusion: Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18:1 do not appear to have a significant effecton the blood lipid profile.

 

2.68           Influence of an algal triacylglycerol containing docosahexaenoic acid (22: 6n-3) and docosapentaenoic acid (22: 5n-6) on cardiovascular risk factors in healthy men and women

Sanders, T.A.B., Gleason, K., Griffin, B. and Miller, G.J.

Br. J. Nutrition, 95, 525-531 (2006)

 

The intake of long-chain n-3 PUFA, including DHA (22: 6n-3), is associated with a reduced risk of CVD. Schizochytrium sp. are an important primary source of DHA in the marine food chain but they also provide substantial quantities of the n-6 PUFA docosapentaenoic acid (22: 5n-6; DPA). The effect of this oil on cardiovascular risk factors was evaluated using a double-blind randomised placebo-controlled parallel-design trial in thirty-nine men and forty women. Subjects received 4 g oil/d for 4 weeks; the active treatment provided 1[middle dot]5 g DHA and 0[middle dot]6 g DPA. Active treatment increased plasma concentrations of arachidonic acid, adrenic acid, DPA and DHA by 21, 11, 11 and 88 mg/l respectively and the proportions of DPA and DHA in erythrocyte phospholipids by 78 and 27 % respectively. Serum total, LDL- and HDL-cholesterol increased by 0[middle dot]33 mmol/l (7[middle dot]3 %), 0[middle dot]26 mmol/l (10[middle dot]4 %) and 0[middle dot]14 mmol/l (9[middle dot]0 %) compared with placebo (all P<=0[middle dot]001). Factor VII (FVII) coagulant activity increased by 12 % following active treatment (P=0[middle dot]006). There were no significant differences between treatments in LDL size, blood pressure, plasma glucose, serum C-reactive protein, plasma FVII antigen, FVII activated, fibrinogen, von Willebrand factor, tocopherol or carotenoid concentrations, plasminogen activator inhibitor-1, creatine kinase or troponin-I activities, haematology or liver function tests or self-reported adverse effects. Overall, the oil was well tolerated and did not adversely affect cardiovascular risk.

 

2.69           Metformin prevents alcohol-induced liver injury in the mouse: critical role of plasminogen activator inhibitor-1

Bergheim, I. et al

Gastroenterology, 130, 2099-2112 (2006)

 

Background & Aims: The biguanide drug metformin has recently been found to improve steatosis and liver damage in animal models and in humans with nonalcoholic steatohepatitis. Methods: The aim of the present study was to determine whether metformin also prevents steatosis and liver damage in mouse models of acute and chronic alcohol exposure. Results: Acute ethanol exposure caused a >20-fold increase in hepatic lipids, peaking 12 hours after administration. Metformin treatment significantly blunted the ethanol effect by >60%. Although metformin is a known inducer of AMP kinase (AMPK) activity, the hepatoprotective property of metformin did not correlate with activation of AMPK or of AMPK-dependent pathways. Instead, the protective effects of metformin correlated with complete prevention of the upregulation of plasminogen activator inhibitor (PAI)-1 caused by ethanol. Indeed, a similar protective effect against acute alcohol-induced lipid accumulation was observed in PAI-1^−/− mice. Hepatic fat accumulation caused by chronic enteral ethanol feeding was also prevented by metformin or by knocking out PAI-1. Under these conditions, necroinflammatory changes caused by ethanol were also significantly attenuated. Conclusions: Taken together, these findings suggest a novel mechanism of action for metformin and identify a new role of PAI-1 in hepatic injury caused by ethanol.

 

2.70           Historical milestones in measurement of HDL-cholesterol: Impact on clinical and laboratory practice 

Langlois, M.R. and Blaton, V.H.

Clin. Chem. Acta, 369, 168-178 (2006)

 

High-density lipoprotein cholesterol (HDL-C) comprises a family of particles with differing physicochemical characteristics. Continuing progress in improving HDL-C analysis has originated from two separate fields—one clinical, reflecting increased attention to HDL-C in estimating risk for coronary heart disease (CHD), and the other analytical, reflecting increased emphasis on finding more reliable and cost-effective HDL-C assays. Epidemiologic and prospective studies established the inverse association of HDL-C with CHD risk, a relationship that is consistent with protective mechanisms demonstrated in basic research and animal studies. Atheroprotective and less atheroprotective HDL subpopulations have been described. Guidelines on primary and secondary CHD prevention, which increased the workload in clinical laboratories, have led to a revolution in HDL-C assay technology. Many analytical techniques including ultracentrifugation, electrophoresis, chromatography, and polyanion precipitation methods have been developed to separate and quantify HDL-C and HDL subclasses. More recently developed homogeneous assays enable direct measurement of HDL-C on an automated analyzer, without the need for manual pretreatment to separate non-HDL. Although homogeneous assays show improved accuracy and precision in normal serum, discrepant results exist in samples with atypical lipoprotein characteristics. Hypertriglyceridemia and monoclonal paraproteins are important interfering factors. A novel approach is nuclear magnetic resonance spectroscopy that allows rapid and reliable analysis of lipoprotein subclasses, which may improve the identification of individuals at increased CHD risk. Apolipoprotein A-I, the major protein of HDL, has been proposed as an alternative cardioprotective marker avoiding the analytical limitations of HDL-C.

 

2.71           Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway

Wilsie, L.C., Gonzales, M. and Orlando, R.A.

Lipids in Health and Disease, 5(23), 1-14 (2006)

 

Background

Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.

Results

Although LRP in these cells is fully active as established by substantial α₂macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K⁺, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.

Conclusion

These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

 

2.72           Effects of altering the ratio of dietary n–6 to n–3 fatty acids on insulin sensitivity, lipoprotein size, and postprandial lipemia in men and postmenopausal women aged 45–70 y: the OPTILIP Study

Griffin, M.D. et al

Am. J. Clin. Nutr., 84, 1290-1298 (2006)

 

Background: Insulin resistance is associated with elevated plasmatriacylglycerol, low HDL concentrations, elevated postprandiallipemia, and a predominance of small, dense LDLs (sdLDLs). Ithas been hypothesized that the dietary ratio of n–6 ton–3 (n–6:n–3) polyunsaturated fatty acids(PUFAs) may have favorable effects on these risk factors byincreasing insulin sensitivity.

Objective: The objective was to measure changes in insulin sensitivity,lipoprotein size, and postprandial lipemia after a 6-mo alterationin n–6:n–3.

Design: In a randomized, parallel design in 258 subjects aged45–70 y, we compared 4 diets providing 6% of energy asPUFAs with an n–6:n–3 between 5:1 and 3:1 with acontrol diet that had an n–6:n–3 of 10:1. The dietswere enriched in -linolenic acid, eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA), or both. Insulin sensitivitywas assessed with the homeostatic model assessment of insulinresistance and the revised quantitative insulin sensitivitytest.

Results: Dietary intervention did not influence insulin sensitivityor postprandial lipase activities. Fasting and postprandialtriacylglycerol concentrations were lower, and the proportionof sdLDLs decreased (by 12.7%; 95% CI: –22.9%, 2.4%),with an n–6:n–3 of 3:1, which was achieved by theaddition of long-chain n–3 PUFAs (EPA and DHA).

Conclusions: Decreasing the n–6:n–3 does not influence insulin sensitivity or lipase activities in older subjects. The reduction in plasma triacylglycerol after an increased intake of n–3 long-chain PUFAs results in favorable changes in LDL size.

 

2.73           Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated by vitamin C in Type 2 diabetes

Anderson, R.A., Evans, L.M., Ellis, G.R., Khan, N., Morrist, K., Jackson, S.K., Rees, A., Lewis, M.J. and Frenneaux, M.P.

Diabetic Med., 2383), 258-264 (2006)

Background  Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C.

Methods  Twenty subjects with T2DM with moderate glycaemic control (mean HbA_1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery.

Results  There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 ± 1.3% to 4 h 0.22 ± 1.1% (P < 0.05) and 8 h 0.7 ± 0.9% (P < 0.05) (mean ± sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 ± 0.9–2.8 ± 0.8 (at 4 h) and 2.9 ± 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 ± 118 (B) to 224.7 ± 72 P < 0.05] and not at 8 h 301.1 ± 118 (B) to 260 ± 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 ± 1 to 2.7 ± 1 at 4 h and 1.95 ± 1.2 at 8 h (P = 0.0002 and 0.33, respectively).

Conclusion  PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.

 

2.74           Novel Porphyrin Conjugates with a Potent Photodynamic Antitumor Effect: Differential Efficacy of Mono- and Bis-β-cyclodextrin Derivatives

Kralova, J., Synytsya, A., Pouckova, P., Koc, M., Dvorak, M. and Kral, V.

Photochem. Photobiol., 82(2), 432-438 (2006)

 

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(β-CD)l exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(β-CD)2. However, in vivo P(β-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm^-2). In contrast, P(β-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(β-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

 

2.75           Gasoline Exhaust Emissions Induce Vascular Remodeling Pathways Involved in Atherosclerosis

Lund, A.K. et al

Toxicol. Sci., 95(2), 485-494 (2007)

 

Epidemiological evidence indicates that environmental air pollutants are positively associated with the development of chronic vascular disease; however, the mechanisms involved have not been fully elucidated. In the present study we examined molecular pathways associated with chronic vascular disease in atherosclerosis-prone apolipoprotein E–deficient (ApoE^–/–) mice, including markers of vascular remodeling and oxidative stress, in response to exposure to the ubiquitous environmental pollutant, gasoline engine emissions. ApoE^–/– mice, on a high-fat diet, were exposed by inhalation to either filtered air; 8, 40, or 60 µg/m³ particulate matter whole exhaust; or filtered exhaust with gases matching the 60-µg/m³ concentration, for 7 weeks. Aortas and plasma were collected and assayed for changes in histochemical markers, real-time reverse transcriptase–polymerase chain reaction, and indicators of oxidative damage. Inhalational exposure to gasoline engine emissions resulted in increased aortic mRNA expression of matrix metalloproteinase-3 (MMP-3), MMP-7, and MMP-9, tissue inhibitor of metalloproteinases-2, endothelin-1 and heme oxygenase-1 in ApoE^–/– mice; increased aortic MMP-9 protein levels were confirmed through immunohistochemistry. Elevated reactive oxygen species were also observed in arteries from exposed animals, despite absence of plasma markers. Similar findings were also observed in the aortas of ApoE^–/– mice exposed to particle-filteredatmosphere, implicating the gaseous components of the wholeexhaust in mediating the expression of markers associated withthe vasculopathy. These findings demonstrate that exposure togasoline engine emissions results in the transcriptional upregulationof factors associated with vascular remodeling, as well as increasedmarkers of vascular oxidative stress, which may contribute tothe progression of atherosclerosis and reduced stability ofvulnerable plaques.

 

2.76           The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

He, J. et al

1. Cell Biol., 176(2), 141-146 (2007)

 

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

 

2.77           Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds 

Vohra, R., Murphy, J.E., Walker, J.H., Homer-Vanniasinkam, S. and Ponnambalam, S.

Protein Expression & Purification, 52(2), 415-421 (2007)

 

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial α-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.

 

2.78           Simultaneous Control of Hyperglycemia and Oxidative Stress Normalizes Endothelial Function in Type 1 Diabetes

Ceriello, A., Kumar, S., Piconi, L., Esposito, K. and Guigliano, D.

Diabetes Care, 30(3), 649-654 (2007)

 

OBJECTIVE—Previous studies have shown that in type 1 diabetesendothelial dysfunction persists even when glycemia is normalized.Moreover, oxidative stress has recently been demonstrated tobe the mediator of hyperglycemia-induced endothelial dysfunction.

RESEARCH DESIGN AND METHODS—Thirty-six type 1 diabeticpatients and 12 control subjects were enrolled. The diabeticpatients were divided into three groups. The first group wastreated for 24 h with insulin, achieving a near-normalizationof glycemia. After 12 h of this treatment, vitamin C was addedfor the remaining 12 h. The second group was treated for 24h with vitamin C. After 12 h of this treatment, insulin wasstarted, with achievement of near-normalization of glycemiafor the remaining 12 h. The third group was treated for 24 hwith both vitamin C and insulin, achieving near-normalizationof glycemia.

RESULTS—Neither normalization of glycemia nor vitaminC treatment alone was able to normalize endothelial dysfunctionor oxidative stress. However, a combination of insulin and vitaminC normalized endothelial dysfunction and decreased oxidativestress to normal levels.

CONCLUSIONS—This study suggests that long-lasting hyperglycemia in type 1 diabetic patients induces permanent alterations in endothelial cells, which may contribute to endothelial dysfunction by increased oxidative stress even when hyperglycemia is normalized.

 

2.79           Secretion of the glucose-regulated selenoprotein SEPS1 from hepatoma cells 

Gao, Y. et al

Biochem. Biophys. Res. Comm., 356, 636-641 (2007)

 

SEPS1 (also called selenoprotein S, SelS, Tanis or VIMP) is a selenoprotein, localized predominantly in the ER membrane and also on the cell surface. In this report, we demonstrate that SEPS1 protein is also secreted from hepatoma cells but not from five other types of cells examined. The secretion can be abolished by the ER-Golgi transport inhibitor Brefeldin A and by the protein synthesis inhibitor cycloheximide. Using a sandwich ELISA, SEPS1 was detected in the sera of 65 out of 209 human subjects (31.1%, average = 15.7 ± 1.1 ng/mL). Fractionation of human serum indicated that SEPS1 was associated with LDL and possibly with VLDL. The function of plasma SEPS1 is unclear but may be related to lipoprotein metabolism.

 

2.80           Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids 

Bateman, P.A., Jackson, K.G., Maitin, V., Yaqoob, P. and Williams, C.M.

Biochim. Biophys. Acta, 1771, 475-485 (2007)

 

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

 

2.81           Large-scale preparation of human low- and high-density lipoproteins by density gradient centrifugation using iodixanol

Billington, D., Maxwell, E., Graham, J.M. and Newland, P.

Anal. Biochem., 367(1), 137-139 (2007)

 

No abstract available

 

2.82           Telmisartan Shows an Equivalent Effect of Vitamin C in Further Improving Endothelial Dysfunction After Glycemia Normalization in Type 1 Diabetes

Ceriello, A., Piconi, L., Esposito, K. and Gugliano, D.

Diabetes Care, 30(7), 1694-1698 (2007)

 

OBJECTIVE— Long-lasting hyperglycemia in type 1 diabeticpatients induces permanent alterations of endothelial functionby increased oxidative stress, even when glycemia is normalized.

RESEARCH DESIGN AND METHODS— In this study, 36 type 1diabetic patients and 12 control subjects were enrolled. Thediabetic patients were divided into three groups. The firstgroup was treated for 24 h with insulin, achieving a near normalizationof glycemia. After 12 h of this treatment, vitamin C was addedfor the remaining 12 h. The second group was treated for 24h with vitamin C. After 12 h of this treatment, insulin wasstarted, achieving a near normalization of glycemia for theremaining 12 h. The third group was treated for 24 h with bothvitamin C and insulin, achieving near normalization of glycemia.The same protocols were performed after 1 month of telmisartanor placebo.

RESULTS— Neither normalization of glycemia nor vitaminC treatment alone was able to normalize endothelial dysfunctionor oxidative stress. Combining insulin and vitamin C normalizedendothelial dysfunction and decreased oxidative stress to normallevels. Telmisartan significantly improved basal endothelialfunction and decreased nitrotyrosine plasma levels. In patientstreated with telmisartan, a near normalization of both flow-mediatedvasodilation and oxidative stress was achieved when glycemiawas normalized, whereas adding vitamin C infusion did not showfurther effect on endothelial function or nitrotyrosine plasmalevels.

CONCLUSIONS— These data indicate that combining the normalizationof glycemia with an antioxidant can normalize endothelial functionin type 1 diabetic patients and that telmisartan works as anantioxidant like vitamin C.

 

2.83           Separation of the principal HDL subclasses by iodixanol gradient ultracentrifugation

Harman, N.L., Davies, I.G. and Griffin, B.A.

Atherosclerosis, 194(1), 283-284 (2007)

 

Introduction: HDL is the smallest and most dense lipoprotein, its main function is in reverse cholesterol transport and as such it can be described as an anti-atherogenic lipoprotein. HDL exists as a number of subclasses differing in size and density; it has been proposed that these subclasses show a variable relationship with CVD risk. The use of HDL subclasses as a CVD risk marker is limited due to the expensive and time consuming nature of current techniques. The aim of the current study was to develop a rapid and cost effective method for the measurement of the principal plasma HDL subclasses.

Methods: HDL subclasses were separated by iodixanol gradient ultracentrifugation (I_xDGUC) from pre-stained plasma, in a run time of 2 h 30 min. A digital image of the separated HDL bands was downloaded and analysed by TotalLab 1D gel scan software to generate HDL subclass profiles. These profiles were validated by co-isolation of the HDL fraction on gradient gel electrophoresis (GGE). HDL was further fractionated and characterised by the distribution of particle size and apo A-1.

Results: Delineation between HDL₂ and HDL₃ was at a density cut-off of 1.059 g/L. There was a significant correlation between HDL subclass for both GGE and I_xDGUC methods as measured by area under the curve [HDL₃ r = 0.77; P < 0.01]. HDL₃% correlated inversely with total HDL-cholesterol [r = −0.64; P < 0.01)].

Conclusion: I_xDGUC provides a rapid, high throughput and cost effective method for the separation of HDL₂ and HDL₃ subclasses.

 

2.84           Characterization of hepatitis C virus associated with very low density lipoprotein (VLDL) in infected human serum and liver

Nielsen, S., Bassendine, M., Neely, D., Ibrahim, S. and Toms, G.

Atherosclerosis, 194(1), 284 (2007)

 

Hepatitis C virus (HCV) particles in serum are complexed with host very low density lipoprotein (VLDL) in lipo-viro particles. These structures are fragile and heterogeneous in density and size but, hitherto, their titres have been too low for detailed structural studies. Our group has previously described a unique liver transplant patient with an unusually high titre of HCV, sufficient for comprehensive biochemical and biophysical characterization.

In order to characterize native HCV lipo-viro particles in this material, serum and liver macerate was first fractionated on iodixanol density gradients. HCV containing fractions with densities below 1.10 g/ml were further characterized by gel filtration, which separates VLDL, LDL and HDL according to size. HCV RNA in serum was found in particles, which co-eluted with large VLDL particles. These are the putative lipo-viro particles and support our earlier findings that HCV circulates in blood in association with VLDL. HCV in liver was found to co-elute with endoplasmic reticulum membranes. However, particles of a similar size to HCV lipo-viro particles in serum contained a high ratio of positive to negative strand HCV RNA suggesting that these fractions are enriched in HCV virus particles associated with VLDL.

 

2.85           Endurance swimming activates trout lipoprotein lipase: plasma lipids as a fuel for muscle

Magnoni, L. and Weber, J-M.

1. Exp. Biol., 210, 4016-4023 (2007)

 

Fish endurance swimming is primarily powered by lipids supplied to red muscle by the circulation, but the mechanism of delivery remains unknown. By analogy to mammals, previous studies have focused on non-esterified fatty acids (NEFA bound to albumin), but lipoproteins have not been considered as an energy shuttle to working muscles. The effects of exercise on fish lipoprotein lipase (LPL) have never been investigated. We hypothesized that LPL and circulating lipoproteins would be modified by prolonged swimming. Because LPL is naturally bound to the endothelium, we have used heparin to release the enzyme in the circulation and to characterize reserve capacity for lipoprotein catabolism. The effects of exercise (4 days at 1.5 body lengths s^–1 in a swim tunnel) were measured for red muscle LPL, post-heparin plasma LPL, and lipoprotein concentration/composition. Red muscle LPL activity increased from 18±5 (rest) to 49± 9 nmol fatty acids min^–1 g^–1 (swimming). In resting fish, heparin administration caused a 27-fold increase in plasma LPL activity that reached a maximum of 1.32± 0.67 µmol fatty acids min^–1 ml^–1 plasma. This heparin-induced response of plasma LPL was not different between resting controls and exercised fish. Heparin or prolonged swimming had no effect on the concentration/composition of lipoproteins that contain 92% of the energy in total plasma lipids. We conclude that (1) red muscle LPL is strongly activated by endurance swimming, (2) rainbow trout have a high reserve capacity for hydrolyzing lipoproteins, and (3) future studies should aim to measure lipoprotein flux because their concentration does not reflect changes in flux. These novel characteristics of fish LPL imply that lipoproteins are used as a metabolic shuttle between fat reserves and working muscles, a strategy exploiting an abundant source of energy in rainbow trout.

 

2.86           Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin

Weeke-Klimp, A.H. et al

1. Drug Targeting, 15(9), 585-594 (2007)

 

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF-SPLPs and untargeted SPLPs labeled with [³H]cholesteryloleyl-ether were injected into rats. About 87% of the LF-SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF-SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF-SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF-SPLPs are not able to transfect these cells in vivo.

 

2.87           Lipoprotein-Heparan Sulfate Interactions in the Hh Pathway

Eugster, C., Panakova, D., Mahmoud, A. and Eaton, S.

Developmental Cell, 13(1), 57-71 (2007)

 

The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Here we show that Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens.

 

2.88           Binding of liver derived, low density hepatitis C virus to human hepatoma cells

Martin, C., Nielsen, S.U., Ibrahim, S., Bassendine, M.F. and Toms, G.L.

1. Med. Virol., 80, 816-823 (2008)

 

HCV recovered from low density fractions of infected blood is associated with lipid and host apo-lipoproteins in lipo-viro-particles (LVP). It has been proposed that these particles are capable of binding and entering hepatocytes by viral glycoprotein independent mechanisms utilizing uptake pathways of normal host lipoproteins after binding to cell surface glycosaminoglycans (GAG), the low density lipoprotein receptor (LDL-r) or scavenger receptor B1 (SR-B1). In this study binding to human hepatoma cells of HCV low density RNA containing particles, semi-purified from macerates of infected human liver, is compared with that of normal host low density lipoprotein (LDL). Binding of both LDL and HCV low density RNA containing particles paralleled LDL-r but not SR-B1 expression on the recipient cells. Binding of both particle types was sensitive to suramin at 0°C but less so at 37°C suggesting that they both bind initially to GAG but, at 37°C, are internalized or transferred to a suramin resistant receptor. Suramin resistant uptake of both particles was blocked in the presence of excess LDL or oxidized LDL. However, whilst LDL uptake was blocked by anti-apoB-100, HCV low density RNA uptake was enhanced by anti-apoB100 and further enhanced by a cocktail of anti-apo-B100 and anti-apoE. Pre-incubation of HCV low density RNA containing particles with antibodies to the E2 glycoprotein had little or no effect on uptake. These data indicate that whilst liver derived HCV RNA containing particles are taken up by HepG2 cells by a virus glycoprotein independent mechanism, the mechanism differs from that of LDL uptake.

 

2.89           TRANSPORT OF GHRELIN AND OBESTATIN IN PLASMA

Holmes, E., Davies, I., Lowe, G. and Ranganath, L.

Atherosclerosis Supplements, 9(1), 26-27 (2008)

 

Introduction: Since it’s discovery in 1999, ghrelin has emerged as a key player in central appetite regulation, and also has cardiovascular actions. Roles for ghrelin in the development of atherosclerosis have also been described. Ghrelin is secreted in an active, acylated form, which is rapidly des-acylated in plasma. Obestatin, a peptide produced from the cleavage of pre-proghrelin, is also emerging as a metabolic regulator. While it has been reported that ghrelin can bind to HDL, knowledge about ghrelin and

obestatin transport and metabolism is limited.

Aims: To investigate the transport of ghrelin and obestatin by lipoproteins.

Methods: Ghrelin and obestatin were measured by EIA in plasma fractions generated using an iodixanol gradient.

Results: Acylated ghrelin (AG) bound to all lipoproteins and was present as a plasma protein (VLDL 12%, LDL 33%, HDL 23%, protein 32% of total AG). In contrast, unacylated ghrelin (UAG) was bound more

specifically to HDL (LDL 5%, HDL 43%, protein 51%). Obestatin did not bind to lipoproteins.

Discussion: AG binding to lipoproteins, may be due to a non-specific, hydrophobic interaction between the acyl group and the phospholipid membrane. The interaction between UAG and HDL may indicate a more

specific interaction; possibly due to des-acylation of ghrelin by a HDL bound enzyme. The implications of these various forms of Ghrelin in the context of obesity and cardiovascular disease is unknown.

 

2.90           Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Yee, M.S. et al

1. Lipid Res., 49, 1364-1371 (2008)

 

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

 

2.91           Phenolsulfonphthalein, but Not Phenolphthalein, Inhibits Amyloid Fibril Formation: Implications for the Modulation of Amyloid Self-Assembly

Levy, M., Porat, Y., Bacharach, E., Shalev, D.E. and Gazit, E.

Biochemistry, 47, 5896-5904 (2008)

 

The study of the mechanism of amyloid fibril formation and its inhibition is of key medical importance due to the lack of amyloid assembly inhibitors that are approved for clinical use. We have previously demonstrated the potent inhibitory potential of phenolsulfonphthalein, a nontoxic compound that was approved for diagnostic use in human subjects, on aggregation of islet amyloid polypeptide (IAPP) that is associated with type 2 diabetes. Here, we extend our studies on the mechanism of action of phenolsulfonphthalein by comparing its antiamyloidogenic effect to a very similar compound that is also approved for human use, phenolphthalein. While these compounds have very similar primary chemical structures, they significantly differ in their three-dimensional conformation. Our results clearly demonstrated that these two compounds had completely different inhibitory potencies: While phenolsulfonphthalein was a very potent inhibitor of amyloid fibril formation by IAPP, phenolphthalein did not show significant antiamyloidogenic activity. This behavior was observed with a short amyloid fragment of IAPP and also with the full-length polypeptide. The NMR spectrum of IAPP₂₀₋₂₉ in the presence of phenolsulfonphthalein showed chemical shift deviations that were different from the unbound or phenolphthalein-bound peptide. Differential activity was also observed in the inhibition of insulin amyloid formation by these two compounds, and density-gradient experiments clearly demonstrated the different inhibitory effect of the two compounds on the formation of prefibrillar assemblies. Taken together, our studies suggest that the three-dimensional arrangement of the polyphenol phenolsulfonphthalein has a central role in its amyloid formation inhibition activity.

 

2.92           Increased dietary cholesterol does not increase plasma low density lipoprotein when accompanied by an energy-restricted diet and weight loss

Harman, N.L., Leeds, A.R. and Griffin, B.A.

Eur. J. Nutr., 47, 287-293 (2008)

 

Background  Diets enriched with dietary cholesterol, frequently from eggs, have been shown to produce a small but variable increase in plasma low density lipoprotein (LDL) cholesterol. There is evidence to suggest that energy-restricted diets, that may contain a relatively high proportion of fat and cholesterol, can attenuate the cholesterol-raising effect of dietary cholesterol on plasma LDL.

Aim of the study  To determine the combined effects of increased dietary cholesterol and weight loss produced by energy restriction on plasma LDL cholesterol and lipoproteins.

Methods  A randomized, controlled, parallel study was performed in two groups of free-living volunteers on an energy-restricted diet for 12 weeks, one group was instructed to consume two eggs a day (n = 24), the other, to exclude eggs (n = 21). Dietary advice on energy restriction was based on the British Heart Foundation guidelines on how to lose weight for men and women.

Results  Energy intake fell by 25 and 29% in the egg-fed and non-egg-fed groups, resulting in a moderate weight loss of 3.4 kg (P < 0.05) and 4.4 kg (P < 0.05), respectively. The daily intake of dietary cholesterol increased significantly in the egg-fed group from 278 to 582 mg after 6 weeks. The concentration of plasma LDL cholesterol decreased in the non-egg-fed groups after 6 weeks (P < 0.01) and in the egg-fed and non-egg-fed at 12 weeks relative to baseline. There were no other significant changes in plasma lipoproteins or LDL particle size.

Conclusions  An increased intake of dietary cholesterol from two eggs a day, does not increase total plasma or LDL cholesterol when accompanied by moderate weight loss. These findings suggest that cholesterol-rich foods should not be excluded from dietary advice to lose weight on account of an unfavorable influence on plasma LDL cholesterol.

 

2.93           The Potential for β-Structure in the Repeat Domain of Tau Protein Determines Aggregation, Synaptic Decay, Neuronal Loss, and Coassembly with Endogenous Tau in Inducible Mouse Models of Tauopathy

Mocanu, M-M. et al

1. Neurosci., 28(3), 737-748 (2008)

 

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimer's disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation K280 (Tau_RD/ K280), or the K280 plus two proline mutations in the hexapeptide motifs (Tau_RD/ K280/I277P/I308P). The K280 mutation accelerates aggregation ("proaggregation mutant"), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models ("antiaggregation mutant"). The inducible transgene expression was driven by the forebrain-specific CaMKII (calcium/calmodulin-dependent protein kinase II ) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2–3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant Tau_RD coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation Tau_RD is switched off, soluble and aggregated exogenous Tau_RD disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.  

 

2.94           mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

Apcher, S., Komarova, A., Daskalogianni, C., Yin, Y., Malbert-Colas, L. And Fåhraes, R.

1. Virol., 83(3), 1289-1298 (2009)

 

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5' untranslated region (UTR) of the c-myc mRNA to the 5' UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

 

 

2.95           Flow-mediated vasodilatation: variation and interrelationships with plasma lipids and lipoproteins

Rasmussen, J.G., Eschen, R.B., Aardestrup, I.V., Dethlefsen, C., Griffin, B.A. and Schmidt, E.B.

Scand. J. Clin. Lab. Invest., 69(1), 156-160 (2009)

 

Objective . Endothelial dysfunction is a critical, prerequisite step in atherosclerosis, and may be evaluated by flow-mediated vasodilatation (FMD). The objective of this study was to examine interrelationships between FMD and plasma lipids and lipoproteins, and to determine the between-operator and within-subject variability associated with this technique. Material and methods. FMD, plasma lipids and lipoproteins, including small dense LDL (sdLDL), were measured twice in 40 healthy volunteers, 4 weeks apart. Interrelationships between mean FMD responses and plasma lipids and lipoproteins were examined by correlation analysis. FMD measurements were taken by two independent operators, allowing determination of between-operator variability. Within-subject variability was determined by obtaining two measurements, 4 weeks apart, in every subject, and carried out by the same operator. Results. FMD was inversely related to plasma triglycerides (r = -0.47, p = 0.002), total cholesterol/HDL cholesterol (r = -0.35, p = 0.03) and apolipoprotein B (r = -0.36, p = 0.02), but not to other plasma lipids and lipoproteins. When measuring variation in FMD, the following results were found: Between operators (SD = 4.0 FMD%) and within subjects (SD = 2.9 FMD%). Conclusions. The associations between FMD, plasma triglycerides and apoB provide evidence supporting a role for triglyceride-rich lipoproteins in endothelial dysfunction.

 

2.96           Phosphotyrosine-dependent in vitro reconstitution of recombinant LAT-nucleated multiprotein signalling complexes on liposomes

Sangani, D., Venien-Bryan, C. and Harder, T.

Mol. Med. Biol., 26(2), 159-170 (2009)

 

Numerous cell surface receptors propagate activation signals to the interior of the cell via tyrosine phosphorylation of transmembrane proteins. This leads to the phosphotyrosine (PiY)-mediated recruitment of cytoplasmic signalling protein complexes which catalyze crucial biochemical signalling reactions. Here we describe the first in vitro reconstitution of such PiY-nucleated protein complexes on an artificial lipid membrane. A tyrosine phosphorylated recombinant variant of the transmembrane adaptor protein Linker for Activation of T cells (PiYLAT) was anchored in liposomes. These PiYLAT proteoliposomes specifically recruited cooperative high avidity signalling protein complexes from Jurkat cytosol. Nucleation of signalling protein assemblies readily occurred on PiYLAT liposomes composed of phosphatidylserine, but not on PiYLAT liposomes composed of phosphatidylcholine. Purified recombinant grb2 alone did not stably associate with tyrosine phosphorylated LAT proteoliposomes. However, when grb2 was presented to the PiYLAT proteoliposomes in the context of Jurkat cytosol it was incorporated into multiprotein signalling complexes. Together the data suggest that these reconstituted high-avidity signalling protein complexes represent a cooperative protein network. This novel in vitro approach offers a novel technology permitting biochemical, structural, and pharmacological analyses of plasma membrane receptor signalling complexes.

 

2.97           The scavenger receptor CD36 plays a role in cytokine-induced macrophage fusion

Helming, L.,Winter, J. and Gordon, S.

1. Cell Sci., 122, 453-459 (2009)

 

Multinucleated giant cells, characteristic of granulomatous infections, originate from the fusion of macrophages. Using an antibody screening strategy we found that the scavenger receptor CD36 participates in macrophage fusion induced by the cytokines IL-4 and GM-CSF. Our results demonstrate that exposure of phosphatidylserine on the cell surface and lipid recognition by CD36 are required for cytokine-induced fusion of macrophages. We also show that CD36 acts in a heterotypic manner during giant-cell formation and that the formation of osteoclasts is independent of CD36. The discovery of molecules involved in the formation of multinucleated giant cells will enable us to determine their functional significance. Furthermore, our results suggest that lipid capture by cell surface receptors may be a general feature of cell fusion.

 

2.98           Loss of Modifier of Cell Adhesion Reveals a Pathway Leading to Axonal Degeneration

Chen, Q., Peto, C.A., Shelton, G.D., Mizisin, A., Sawchenko, P.E. and Schubert, D.

1. Neurosci., 29(1), 118-130 (2009)

 

Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration.

 

2.99           DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

Wang, H., Kim, S.. and Ryu, W-S.

1. Virol., 83(11), 5815-5824 (2009)

 

Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.

 

2.100           Photoinitiated Destruction of Composite Porphyrin−Protein Polymersomes

Robbins, G.P., Jimbo, M., Swift, J., Therien, M.J., Hammer, D.A. and Dmochoxski, I.J.

1. Am. Chem. Soc., 131, 3872-3874 (2009)

 

Bilayer vesicles assembled from amphiphilic diblock copolymers (polymersomes) adopt asymmetric structures when loaded with moderate concentrations (≥1.5 mg/mL) of horse spleen ferritin (HSF) or its iron-free variant (HSAF). Incorporation of both ferritin and a zinc porphyrin dimer (PZn₂) generates photoresponsive vesicles: irradiation with focused light of near-UV to near-IR wavelengths induces polymersome deformation and destruction on the minute time scale. To investigate this phenomenon, polymersomes were loaded with dye-labeled ferritin and PZn₂. Confocal microscopy identified BODIPY-FL-labeled ferritin at the membrane, whereas Cy3-labeled ferritin was found both at the membrane and throughout the aqueous core. Fluorescence recovery after photobleaching (FRAP) experiments confirmed that Cy3- and BODIPY-FL-labeled ferritin and PZn₂ exhibited slow diffusion at the membrane, consistent with membrane association. Furthermore, micropipette aspiration experiments revealed increased elastic moduli and altered bending rigidity in vesicles incorporating HSAF. Finally, a small molecule (biocytin) was encapsulated within the ferritin−PZn₂ vesicles and released upon exposure to light. These data indicate synergy between ferritin, whose membrane association lowers the barrier to deformation, and PZn₂, which embeds in the membrane, harvests light energy and produces local heating that may lead to membrane budding. This appears to be a general protein−polymer membrane phenomenon, as replacement of ferritin with bovine serum albumin or equine skeletal myoglobin resulted in vesicles with similar asymmetric morphology and photosensitivity.

 

2.101           Four Conserved Cysteine Residues of the Hepatitis B Virus Polymerase Are Critical for RNA Pregenome Encapsidation

Kim, S., Lee, J. and Ryu, W-S.

1. Virol., 83(16), 8032-8040 (2009)

 

Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.

 

2.102           Effect of hyperthermia on plasma lipids and gene expression in Atlantic Cod. (Gadus Morhua I.)

Aursnes, I.A.S., Gjoen, T. and Rishovd, A-L.

Toxico. Lett., 189S, S57 (2009)

 

Purpose: Wild fish occupy their preferred temperature range by vertical movements in the water column. During fish farming, this migration may be restricted leading to thermal stress. We have investigated stress responses in Atlantic Cod (Gadus Morhua) exposed to elevated water temperature. The main goal was to identify stable housekeeping genes that can be used for internal normalization for quantitative real-time PCR analyses across treatment groups in experiments. The software used was Normfinder and REST. We also obtained blood samples and analyzed for differences in plasma lipid concentrations.

Methods: Wild Atlantic cod (Gadus Morhua) weighing 200–500 g was divided into two groups (5 fish in each tan were one served as control (kept at 12 °C) and one group in which the water temperature was increased from 12 °C to 17 °C. Tissue samples and blood samples were withdrawn from all individuals. Blood was analyzed by separation on a continuous gradient using iodixanol and divided into fractions which was analyzed for triglycerides, cholesterol and level of oxidation. The tissue samples were prepared for RNA extraction and RT-qPCR analyses.

Results: Plasma lipid concentrations (total cholesterol and triglycerides), and oxidation levels in the hyperthermally stressed fish were significantly increased compared to the control group. Across both groups and all tissues Normfinder ranked the gene analyzed in this order, from most stable to least stable EF1α > 18s > ubiquitin > β-actin > β-2-microglobulin > tubulin α > ARP > G6PDH. In liver tissue Normfinder identified ubiquitin as the most stable gene. REST analysis of relative expression revealed that in liver HSP90, IL-1β and TNFα was significantly upregulated after hyperthermia.

 

2.103           Comparability of methods for LDL subfraction determination: A systematic review

Chung, M., Lichtenstein, A.H., Ip, S., Lau, J. and Balk, E.M.

Atherosclerosis, 205, 342-348 (2009)

 

Identifying and aggressively treating individuals at elevated risk of developing cardiovascular disease (CVD) is critical to optimizing health outcomes. The CVD risk factors defined by the National Cholesterol Education Program do not fully predict individuals at high risk of developing CVD. Validation of potential methodologies against a reference method is essential to the adoption of a potential new risk factor to improve risk prediction. Low-density lipoprotein (LDL) subfraction has been advanced as a potential additional CVD risk factor. Currently, there is no reference method for determining LDL subfractions or standardizing the different methods used to measure LDL subfractions. We conducted a systematic review to identify reports comparing two or more methods of measuring LDL subfractions. Nine articles were identified that separated and quantified LDL subfractions by at least two methods. Comparative data were available for nuclear magnetic resonance vs. gel electrophoresis (GE), LipoPrint^® vs. other GE methods, ultracentrifugation vs. GE, and high performance gel filtration chromatography vs. GE. We found a wide range of agreement (from 7 to 94% concordance for classifying LDL patterns) among methods for LDL subfraction determinations. Different criteria and definitions were used among the articles to classify individuals with respect to CVD risk. No study used CVD or other clinical outcomes as an outcome measure. In summary, the currently available literature does not provide adequate data about comparability in terms of test performance to choose one or another method to serve as a standard nor are data on comparability in terms of predicting CVD outcomes.

 

2.104           Changes in lipoprotein profile and urinary albumin excretion in familial LCAT deficiency with lipid lowering therapy

Yee, M.S., Pavitt, D.V., Richmond, W., Cook, H.T., McLean, A.G., Valabhji, J. and Elkeles, R.S.

Atherosclerosis, 205, 528-532 (2009)

 

Familial lecithin:cholesterol acyltransferase deficiency (FLD) is a monogenic autosomal recessive condition, affecting cholesterol esterification and leads to progressive renal impairment and end-stage renal failure, probably due to the abnormal lipoprotein (X) (Lp(X)).

We report a case of FLD, whom we treated with a combination of nicotinic acid 1.5 g nocte and fenofibrate M/R 160 mg od and report changes in lipid profile and Lp(X), after six weeks and serum creatinine and urine albumin/creatinine ratio after 12 months. We assessed the cardiovascular risk using electron beam computed tomography.

At baseline total cholesterol was 6.61 mmol/L; HDL cholesterol 0.57 mmol/L; Lp(X) cholesterol 3.24 mmol/L; triglyceride 4.13 mmol/L; apolipoprotein A1 46 mg/dL; and apolipoprotein B 53 mg/dL. After six weeks of treatment his total cholesterol was 4.16; HDL cholesterol 0.52; Lp(X) cholesterol 1.73 mmol/L; triglyceride 1.80 mmol/L; apolipoprotein A1 36 mg/dL; and apolipoprotein B 50 mg/dL. Baseline serum creatinine was 106 μmol/L and urine albumin/creatinine ratio was 127.3 mg/mmol and after 12 months was 101 μmol/L and 31.5 mg/mmol respectively. His coronary artery calcification score was zero.

We have shown, we believe for the first time, that combination lipid modifying therapy in FLD leads to a reduction in Lp(X) concentration and an associated reduction in urine albumin excretion at 12 months.

 

2.105           Long-Term Glycemic Control Influences the Long-Lasting Effect of Hyperglycemia on Endothelial Function in Type 1 Diabetes

Ceriello, A., Esposito, K., Ihnat, M., Thorpe, J. and Giugliano, D.

1. Clin. Endocrinol. Metab., 94(8), 2751-2756 (2009)

 

Objective: The objective of the study was to investigate theeffect of different periods of hyperglycemia on the reversalof endothelial dysfunction by glucose normalization and antioxidanttherapy.

Research Design and Methods: Ten healthy subjects and threesubgroups of 10 type 1 diabetic subjects were enrolled as follows:1) patients within 1 month of diagnosis; 2) patients between4.5 and 5.2 yr from diagnosis and with glycosylated hemoglobinlevels 7% or greater since diagnosis; 3) patients between 4.8and 5.4 yr from diagnosis and with glycosylated hemoglobin levelsgreater than 7% since diagnosis. Each patient participated inthree experiments: 1) 24-h insulin treatment, achieving a nearnormalization of glycemia, together with the addition of theantioxidant vitamin C during the last 12 h; 2) 24-h vitaminC treatment with insulin treatment for the last 12 h; and 3)treatment with both vitamin C and insulin for 24 h.

Results: Endothelial function, as measured by flow-mediatedvasodilation of the brachial artery and levels of nitrotyrosine,an oxidative stress marker, were normalized by each treatmentin subgroups 1 and 2. In the third subgroup, neither glucosenormalization nor vitamin C treatment alone was able to normalizeendothelial dysfunction or oxidative stress. Combining insulinand vitamin C, however, normalized endothelial dysfunction andnitrotyrosine.

Conclusions: This study suggests that long-lasting hyperglycemiain type 1 diabetic patients induces long-term alterations inendothelial cells, which may contribute to endothelial dysfunctionand is interrupted only by both glucose and oxidative stressnormalization.

 

2.106           The accessory subunit of mitochondrial DNA polymerase determines the DNA content of mitochondrial nucleoids in human cultured cells

Re, M.D., Sembongi, H., He, J., Reyes, J.H., Yasukawa, T., Martinsson, P., Bailey, L.J., Goffart, S., Boyd-Kirkup, J.D., Wong, T.S., Fersht, A.R., Spelbrink, J.N. and Holt, I.J.

Nucleic Acids Res., 37(17), 5701-5713 (2009)

 

The accessory subunit of mitochondrial DNA polymerase , POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLG . These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

 

2.107           Ultrastructures and strain comparison of under-glycosylated scrapie prion fibrils

Sim, V.L. and Caughey, B.

Neurobiology of Aging, 30, 2031-2042 (2009)

 

Prions, composed primarily of misfolded, often fibrillar, polymers of prion protein, have poorly understood structures. Heavy surface glycosylation may obscure visualization of their fibrillar cores, so we purified severely under-glycosylated prion protein fibrils from scrapie-infected transgenic mice expressing anchorless prion protein. Using electron and atomic force microscopy, we obtained dimensions and morphological information about prion protein core protofilaments which variably intertwined to form scrapie fibrils. Occasional isolated protofilaments were observed, suggesting that the lateral association of protofilaments is neither essential nor invariant in prion protein polymerization. Strain comparisons suggested basic structural differences; ME7 and 22L fibrils contained thinner protofilaments, 22L fibrils preferred left-handed twists, and 22L fibril periodicities averaged 106 nm per half-turn, compared with 64 and 66 nm for RML and ME7 fibrils, respectively. The strains displayed overlapping fibril morphologies, providing evidence that prion fibril morphology is influenced, but not dictated, by strain-dependent differences in protofilament structure. These measurements of the amyloid core of scrapie fibrils should aid development of models of prion structure and strain determination.

 

2.108           Phytosterol-Enriched Yogurt Increases LDL Affinity and Reduces CD36 Expression in Polygenic Hypercholesterolemia

Ruiu, G., Pinach, S., Veglia, F., Gambino, R., Marena, S., Uberti, B.., Alemanno, N., Burt, D., Pagano, G. and Cassader, M.

Lipids, 44, 153-160 (2009)

 

Dietary enrichment with phytosterols (plant sterols similar to cholesterol) is able to reduce plasma cholesterol levels due to reduced intestinal absorption. The aim of this study was to investigate the effect of phytosterol-enriched yogurt consumption on the major serum lipid parameters, low density lipoprotein (LDL) receptor activity, LDL-receptor affinity, and CD36 expression in hypercholesterolemic subjects. Fifteen patients affected by polygenic hypercholesterolemia were evaluated in a single-blind randomized crossover study after a 4 weeks treatment with a phytosterol-enriched yogurt containing 1.6 g esterefied phytosterols (equivalent to 1.0 g free phytosterol). Lipid parameters were compared with a phytosterol-free placebo-controlled diet. The effect of the two treatments on each variable, measured as percentage change, was compared by paired samples t test and covariance analysis. The treatment induced a modest but significant decrease in LDL-cholesterol levels (4.3%, P = 0.03) and a significant increase in high density lipoprotein (HDL) 3-cholesterol (17.1%, P = 0.01). Phytosterol consumption had no effect on LDL-receptor activity whereas patient LDL-receptor affinity significantly increased (9.7%, P = 0.01) and CD36 expression showed a marked significant decrease (18.2%, P = 0.01) in the phytosterol-enriched yoghurt patients. Our data show that the oral administration of a phytosterol-enriched yogurt has modest but significant effects on commonly measured lipid parameters. The improvement of LDL-receptor affinity and the reduction in CD36 expression may reflect an important antiatherogenic effect.

 

2.109           Circulating ghrelin exists in both lipoprotein bound and free forms

Holmes, E., Davies, I., Lowe, G. and Ranganath, L.R.

Ann. Clin. Biochem., 46, 514-516 (2009)

 

Introduction: Ghrelin is a gastric peptide that has been implicated in thedevelopment of obesity and cardiovascular disease. It has beenreported that ghrelin binds to lipoproteins, although the differentbinding patterns of acylated ghrelin (AG) and unacylated ghrelin(UAG) are still to be determined.

Methods: Lipoprotein fractions were generated using a self-generating iodixanol gradient. AG and UAG were measured using specificenzyme immunoassays.

Results: AG bound to all lipoproteins in approximately equal concentrations(VLDL 26%, LDL 22%, HDL 23%) and was present as a plasma protein(27%). UAG bound more specifically to HDL (49%) and was presentas a plasma protein (48%).

Conclusions: The different binding patterns of AG and UAG may have significant implications for their biological effects, including roles in energy metabolism, the development of obesity and potentially in the modulation of cardiovascular disease.

 

2.110           Impact of Saturated, Polyunsaturated and Monounsaturated Fatty Acid-Rich Micelles on Lipoprotein Synthesis and Secretion in Caco-2

Jackson, K.G., Bateman, P.A., Yaqoob, P. and Williams, C.M.

Lipids, 44, 1081-1089 (2009)

 

Meal fatty acids have been shown to modulate the size and composition of triacylglycerol (TAG)-rich lipoproteins influencing the magnitude and duration of the postprandial plasma TAG response. As a result there is considerable interest in the origin of these meal fatty-acid induced differences in particle composition. Caco-2 cells were incubated over 4 days with fatty acid mixtures resembling the composition of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA)-rich meals fed in a previous postprandial study to determine their impact on lipoprotein synthesis and secretion. The MUFA- and PUFA-rich mixtures supported greater intracellular TAG, but not cholesterol accumulation compared with the SFA-rich mixture (P < 0.001). The MUFA-rich mixture promoted significantly greater TAG and cholesterol secretion than the other mixtures and significantly more apolipoprotein B-100 secretion than the PUFA-rich mixture (P < 0.05). Electron microscopy revealed the SFA-rich mixture had led to unfavourable effects on cellular morphology, compared with the unsaturated fatty acid-rich mixtures. Our findings suggest the MUFA-rich mixture, may support the formation of a greater number of TAG-rich lipoproteins, which is consistent with indirect observations from our human study. Our electron micrographs are suggestive that some endocytotic uptake of MUFA-rich taurocholate micelles may promote greater lipoprotein synthesis and secretion in Caco-2 cells.

 

2.111           Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

Kim, S., Wang, H. and Ryu, W-S.

1. Virol., 84(1), 52-58 (2010)

 

The DNA genome of hepatitis B virus (HBV) replicates via reverse transcription within capsids following the encapsidation of an RNA template, the pregenomic RNA (pgRNA). We previously demonstrated that the 5' cap proximity of the stem-loop structure ( or epsilon), an encapsidation signal, is critically important for the encapsidation of the pgRNA (J. K. Jeong, G. S. Yoon, and W. S. Ryu, J. Virol. 74:5502-5508, 2000). Therefore, we speculated that the viral polymerase (Pol), while bound to the 5' stem-loop structure, could recognize the cap via its interaction with eIF4E, a eukaryotic translation initiation factor. Our data showed the direct interaction between HBV Pol and eIF4E, as measured by coimmunoprecipitation. Further, we demonstrated that eIF4E interacts with the Pol- ribonucleoprotein complex (RNP) rather than Pol alone, resulting in eIF4E-Pol- RNP complex formation. In addition, we asked whether eIF4E remains engaged to the Pol- RNP complex during nucleocapsid assembly. Density gradient analysis revealed that eIF4E indeed was incorporated into nucleocapsids. It is of great importance to uncover whether the incorporated eIF4E contributes to viral reverse transcription or other steps in the HBV life cycle.

 

2.112           Unpacking a gel-forming mucin: a view of MUC5B organization after granular release

Kesimer, M., Makhov, A.M., Griffith, J.D., Verdugo, P. and Sheehan, J.K.

Am. J. Physiol. Lung Cell Mol. Physiol., 298, L15-L22 (2010)

 

Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. It has an average mass of 150 x 10⁶ Da and size Rg of 150 nm in radius of gyration. In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH₂- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release.  

 

2.113           Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Dandler, J., Wilhelm, B. and Scheer, H.

Photochem. and Photobiol., 86, 182-193 (2010)

 

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.

 

2.114           Acrolein consumption induces systemic dyslipidemia and lipoprotein modification

Conklin, D.J., Barski, O.A., Lesgards, J-F., Juvan, P., rezen, T., Rozman, D., Prough, R.A., Vladykovskaya, E., Liu, S., Srivastava, S. and Bhatnagar, A.

Tox. Appl. Pharmacol., 243(1), 1.12 (2010)

 

Aldehydes such as acrolein are ubiquitous pollutants present in automobile exhaust, cigarette, wood, and coal smoke. Such aldehydes are also constituents of several food substances and are present in drinking water, irrigation canals, and effluents from manufacturing plants. Oral intake represents the most significant source of exposure to acrolein and related aldehydes. To study the effects of short-term oral exposure to acrolein on lipoprotein levels and metabolism, adult mice were gavage-fed 0.1 to 5 mg acrolein/kg bwt and changes in plasma lipoproteins were assessed. Changes in hepatic gene expression related to lipid metabolism and cytokines were examined by qRT-PCR analysis. Acrolein feeding did not affect body weight, blood urea nitrogen, plasma creatinine, electrolytes, cytokines or liver enzymes, but increased plasma cholesterol and triglycerides. Similar results were obtained with apoE-null mice. Plasma lipoproteins from acrolein-fed mice showed altered electrophoretic mobility on agarose gels. Chromatographic analysis revealed elevated VLDL cholesterol, phospholipids, and triglycerides levels with little change in LDL or HDL. NMR analysis indicated shifts from small to large VLDL and from large to medium-small LDL with no change in the size of HDL particles. Increased plasma VLDL was associated with a significant decrease in post-heparin plasma hepatic lipase activity and a decrease in hepatic expression of hepatic lipase. These observations suggest that oral exposure to acrolein could induce or exacerbate systemic dyslipidemia and thereby contribute to cardiovascular disease risk.

 

2.115           Comparative proteomic profiling of plasma very-low-density and low-density lipoproteins

Sun, H-Y., Chen, S-F., Lai, M-D., Chang, T-T., Chen, T-L., Li, P-Y., Shieh, D-B. and Young, K-C.

Clin. Chim. Acta., 411, 336-344 (2010)

 

Background

Low-density lipoprotein (LDL) is a natural metabolite of very-low-density lipoprotein (VLDL) in the circulation. Systematic investigation of total protein components and dynamics might provide insights into this normal metabolic process.

Methods

VLDL and LDL were purified from normolipidemia pooled plasma by gradient ultracentrifugation with either ionic or non-ionic media. The protein contents were compared by liquid chromatography tandem mass analyses based on isobaric tag for relative and absolute quantitation and two-dimensional gel electrophoresis.

Results

Our comparative lipoproteomes revealed 21 associated proteins. Combined with Western blot analysis, and on the basis of the differential expression levels we classified them into 3 groups: (i) VLDL > LDL [apolipoprotein (apo) A-IV, apo(a), apoCs, apoE, apoJ and serum amyloid A-4]; (ii) VLDL < LDL [albumin, α-1-antitrypsin, apoD, apoF, apoM, and paraoxonase-1]; and (iii) VLDL = LDL [apoA-I, apoA-II, apoB-100, apoL-I and prenylcysteine oxidase-1]. The apoA-I level positively correlated with PCYOX1 but negatively with apoM in VLDL and LDL. Furthermore, the two-dimensional maps displayed 5 apoA-I isoforms in which phosphorylation at Ser55, Ser166, Thr185, Thr221 and Ser252 residues were identified.

Conclusions

This study revealed the VLDL- and LDL lipoproteomes and the full-spectrum protein changes during physiological VLDL-to-LDL transition. It provides a valuable dataset VLDL and LDL proteomes potentially applied to the development of diagnostics.

 

2.116           Isolation and Characterization of Cytoplasmic Cofilin-Actin Rods

Minamide, L.S., Maiti, S., Boyle, J.A., Davis, R.C., Coppinger, J.A., Bao, Y., Huang, T.Y., yates, J., Bokoch, G.M. and Bamburg, J.R.

1. Biol. Chem., 285(8), 5450-5460 (2010)

 

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca²⁺, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

 

2.117           Increased Glycation and Oxidative Damage to Apolipoprotein B100 of LDL Cholesterol in Patients With Type 2 Diabetes and Effect of Metformin

Rabbani, N., Chittari, M.V., Bopdmer, C.W., Zehnder, D., Ceriello, A. and Thornalley, P.J.

Diabetes, 59, 1038-1045 (2010)

 

OBJECTIVE The aim of this study was to investigate whether apolipoprotein B100 of LDL suffers increased damage by glycation, oxidation, and nitration in patients with type 2 diabetes, including patients receiving metformin therapy.

RESEARCH DESIGN AND METHODS For this study, 32 type 2 diabetic patients and 21 healthy control subjects were recruited; 13 diabetic patients were receiving metformin therapy (median dose: 1.50 g/day). LDL was isolated from venous plasma by ultracentrifugation, delipidated, digested, and analyzed for protein glycation, oxidation, and nitration adducts by stable isotopic dilution analysis tandem mass spectrometry.

RESULTS Advanced glycation end product (AGE) content of apolipoprotein B100 of LDL from type 2 diabetic patients was higher than from healthy subjects: arginine-derived AGE, 15.8 vs. 5.3 mol% (P < 0.001); and lysine-derived AGE, 2.5 vs. 1.5 mol% (P < 0.05). Oxidative damage, mainly methionine sulfoxide residues, was also increased: 2.5 vs. 1.1 molar equivalents (P < 0.001). 3-Nitrotyrosine content was decreased: 0.04 vs. 0.12 mol% (P < 0.05). In diabetic patients receiving metformin therapy, arginine-derived AGE and methionine sulfoxide were lower than in patients not receiving metformin: 19.3 vs. 8.9 mol% (P < 0.01) and 2.9 vs. 1.9 mol% (P < 0.05), respectively; 3-nitrotyrosine content was higher: 0.10 vs. 0.03 mol% (P < 0.05). Fructosyl-lysine residue content correlated positively with fasting plasma glucose. Arginine-derived AGE residue contents were intercorrelated and also correlated positively with methionine sulfoxide.

CONCLUSIONS Patients with type 2 diabetes had increased arginine-derived AGEs and oxidative damage in apolipoprotein B100 of LDL. This was lower in patients receiving metformin therapy, which may contribute to decreased oxidative damage, atherogenicity, and cardiovascular disease.

 

2.118           The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Tixador, P., Herzog, L., Reine, F., Jaumain, E., chapuis, J., Le Dur, A., Laude, H. and Beringue, V.

PloSPathogens, 6(4), e1000859 (2010)

 

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP^Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

Prions are unconventional transmissible agents causing fatal neurodegenerative diseases in human and animals. They are thought to be formed from polymers of abnormal conformations of the host-encoded prion protein (PrP), but little is known about the physical organization of the infectious particles and any relationship between packing order and infectivity. As an additional layer of complexity, different PrP conformational variants associated with distinct biological phenotypes, or ‘strains’, can propagate in the same host. We subjected PrP polymers from eight different ovine and hamster prion strains to sedimentation velocity centrifugation, which allows separation of macromolecular complexes according to their size, density or shape. We showed that, whereas the PrP sedimentation profiles share common features, the infectivity profiles exhibit striking differences amongst the strains. For four of them, the infectious component was predominantly associated with slowly sedimenting particles, suggestive of small size oligomers and/or low density PrP aggregates. Such particles appeared to be a feature of strains able to induce a rapidly lethal disease in the recipient host. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

 

2.119           The Conserved Bardet-Biedl Syndrome Proteins Assemble a Coat that Traffics Membrane Proteins to Cilia

Jin, H., White, S.R., Shida, T., Schulz, S., Aguiar, M., Gygi, S.P., Bazan, J.F. and Nachury, M.V.

Cell, 141, 1208-1219 (2010)

 

The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6^GTP-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.

 

2.120           Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Di Giovanni, J., Iborra, C., Maulet, Y., Leveque, C., El Far, O. and Seagar, M.

1. Biol. Chem., 285(31), 23665-23675 (2010)

 

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.

 

2.121           Cigarette smoke and human plasma lycopene depletion

Graham, D.L., Carail, M., Caris-Veyrat, C. and Lowe, G.M.

Food and Chem. Tox., 48, 2413-2420 (2010)

 

It is known that smokers have a higher risk of developing cardiovascular disease and lung cancer. Plasma carotenoid concentrations in smokers are generally lower than in non-smokers and this may be due to modifications in diet or a direct or indirect action of cigarette smoke on carotenoids in the plasma. Recently it was reported that reactive nitrogen species derived from cigarette smoke could diffuse across the lung alveolar cell wall into the plasma. Such species may modify circulating low density lipoprotein (LDL) and in the process reduce circulating carotenoid concentrations. In an effort to address this rational we have treated lycopene solutions, human plasma and isolated LDL with cigarette smoke and monitored all-(E)-lycopene, 5(Z)-lycopene and β-carotene depletion. In plasma, the depletion of all-(E)-lycopene (15.0 ± 11.0%, n = 10) was greater than 5(Z)-lycopene (10.4 ± 9.6%) or β-carotene (12.4 ± 10.5%). In LDL, both all-(E)- and 5(Z)-lycopene were more susceptible than β-carotene (20.8 ± 11.8%, 15.4 ± 11.5% and 11.5 ± 12.5%, n = 3 respectively). The effects have been compared with Sin-1 reactions and isomerization of all-(E) lycopene is common to both treatments. The results clearly indicate that low plasma lycopene may be a direct consequence of smoke inhalation.

 

2.122           Lipoprotein Particles Cross the Blood–Brain Barrier in Drosophila

Brankatschk, M. and Eaton, S.

1. Neurosci., 30(31), 10441-10447 (2010)

 

The blood–brain barrier (BBB) regulates passage of nutrients and signaling molecules from the circulation into the brain. Whether lipoproteins cross the BBB in vivo has been controversial, and no clear requirement for circulating lipoproteins in brain development has been shown. We address these issues in Drosophila, which has an functionally conserved BBB, and lipoproteins that resemble those of vertebrates. We show that the Drosophila lipoprotein lipophorin exists in two isoforms. Both isoforms cross the BBB, but accumulate on distinct subsets of cells within the brain. In addition to acting as a lipid carrier, lipophorin carries both sterol-linked and GPI-linked proteins into the circulation and transports them across the BBB. Finally, lipophorin promotes neuroblast proliferation by a mechanism that does not depend on delivery of dietary lipids. Transport of lipophorin and its cargo across the BBB represents a novel mechanism by which peripherally synthesized proteins might enter the brain and influence its development. Furthermore, lipid-linkage may be an efficient method to transport therapeutic molecules across the BBB.

 

2.123           Tunable Leuko-polymersomes That Adhere Specifically to Inflammatory Markers

Robbins, G.P., Saunders, R.L., Haun, J.B., Rawson, J., Therien, M.J. and Hammer, D.A.

Langmuir, 26(17), 14089-14096 (2010)

 

The polymersome, a fully synthetic cell mimetic, is a tunable platform for drug delivery vehicles to detect and treat disease (theranostics). Here, we design a leuko-polymersome, a polymersome with the adhesive properties of leukocytes, which can effectively bind to inflammatory sites under flow. We hypothesize that optimal leukocyte adhesion can be recreated with ligands that mimic receptors of the two major leukocyte molecular adhesion pathways, the selectins and the integrins. Polymersomes functionalized with sialyl Lewis X and an antibody against ICAM-1 adhere avidly and selectively to surfaces coated with inflammatory adhesion molecules P-selectin and ICAM-1 under flow. We find that maximal adhesion occurs at intermediate densities of both sialyl Lewis X and anti-ICAM-1, owing to synergistic binding effects between the two ligands. Leuko-polymersomes bearing these two receptor mimetics adhere under physiological shear rates to inflamed endothelium in an in vitro flow chamber at a rate 7.5 times higher than those to uninflamed endothelium. This work clearly demonstrates that polymersomes bearing only a single ligand bind less avidly and with lower selectivity, thus suggesting proper mimicry of leukocyte adhesion requires contributions from both pathways. This work establishes a basis for the design of polymersomes for targeted drug delivery in inflammation.

 

2.124           Engineering Therapeutic Nanocarriers with Optimal Adhesion for Targeting 

Haun, J.B., Robbins, G.P. and Hammer, D.A.

1. Adhesion, 86, 131-159 (2010)

 

There is considerable interest in developing therapeutic delivery carriers that can be targeted via receptor-ligand interactions to sites within the blood stream. The adhesion of carriers is determined by the combined effects of transport phenomena, hydrodynamic force, and the dynamics of multivalent receptor/ligand bonding. Optimizing the adhesion of carriers requires developing relationships between these factors and carrier properties such as size and receptor coating density. Recently, we developed canonical relationships for the binding of antibody-conjugated 200 nm particles to surfaces coated with a vascular adhesion molecule, intercellular adhesion molecule-1. Here we extend our previous studies of adhesion to particles of different size, including 40 nm and 1  m particles. Particle binding is assessed under fluid flow in a parallel plate flow chamber while varying particle receptor density, substrate ligand density, and flow rate. Using a stochastic simulation and transport-reaction model we then extract multivalent kinetic rate constants for particle attachment and detachment from the binding data. We demonstrate that particles go though a maximum in binding with particle size. For small particles, increasing size increases receptor-ligand encounter rates; for larger particles, fluid shear force begins to dominate, leading to higher forces and decreased adhesion. Our methods provide a means for optimizing particle size and receptor density for the selective binding of particles to vascular endothelium under flow.

 

2.125           Lack of effect of cold water prawns on plasma cholesterol and lipoproteins in normo-lipidaemic men

Isherwood, C., Wong, M., Jones, W.S., Davies, I.G. and Griffin, B.A.

Cell. Mol. Biol., 56(1), 52-58 (2010)

 

OBJECTIVE: Dietary guidelines for the prevention of coronary heart disease (CHD) have restricted the intake of foods rich in dietary cholesterol, on the grounds that the dietary cholesterol will increase blood cholesterol. In the case of shellfish, this recommendation may limit the intake of a valuable dietary source of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA). The objective of this study was to undertake a dietary intervention to determine the effects of cold water prawns on plasma lipids and lipoproteins.

METHODS: 23 healthy male subjects were randomised to receive either 225 g of cold water prawns or an equivalent weight of fish ('crab') sticks as a control for 12 weeks in a cross-over design. Blood samples were taken at the beginning and end of each intervention for the determination of plasma lipids and lipoproteins by routine enzymatic assays and iodixanol density gradient centrifugation respectively.

RESULTS: The diets were well matched for the intake of total energy and macronutrients, and body weight remained stable throughout the study. The prawn intervention increased the intake of dietary cholesterol to 750 mg/d against 200 mg/d on the control. The intake of LC n-3 PUFA from prawns was estimated to be between 0.5-0.7 g/d. The consumption of prawns produced no significant effects on the concentration of plasma total or LDL cholesterol, triacylglycerol, HDL cholesterol or apolipoproteins A-I and B relative to the control, or within each intervention group over time. There was also no significant effect on LDL density (particle size) relative to the control, or any difference between and within treatments in total plasma lipoprotein profiles by density gradient centrifugation.

CONCLUSION: These findings provide evidence to suggest that the consumption of cold water prawns, at least in healthy, male subjects, should not be restricted on the grounds of this seafood producing an adverse effect on plasma LDL cholesterol.

 

2.126           A Generalized System for Photoresponsive Membrane Rupture in Polymersomes

Kamat, N.P., Robbins, G.P., Rawson, J., Therien, M.J., Dmochowski, I.J. and Hammer, D.A.

Adv. Funct. Mater., 20, 2588-2596 (2010)

 

Polymersomes are vesicles whose membranes comprise self-assembled block copolymers. It has recently been shown that co-encapsulating conjugated multiporphyrin dyes in a polymersome membrane with ferritin protein in the aqueous lumen confers photolability to the polymersome. In the present study, the photolability is shown to be extendable to vesicles containing dextran, an inert and inexpensive polysaccharide, as the luminal solute. How structural features of the polymersome/porphyrin/dextran composite affect its photoresponse is explored. Increasing dextran molecular weight, decreasing block copolymer molecular weight, and altering fluorophore-membrane interactions results in increasing the photoresponsiveness of the polymersomes. Amphiphilic interactions of the luminal encapsulant with the membrane coupled with localized heat production in the hydrophobic bilayer likely cause differential thermal expansion in the membrane and the subsequent membrane rupture. This study suggests a general approach to impart photoresponsiveness to any biomimetic vesicle system without chemical modification, as well as a simple, bio-inert method for constructing photosensitive carriers for controlled release of encapsulants.

 

2.127           Lipid accumulation and metabolism in polychaete spermatogenesis: Role of the large discoidal lipoprotein

Schenk, S. and Hoeger, U.

Mol. Reprod. Dev., 77, 710-719 (2010)

 

In most oviparous animals, lipoprotein-mediated lipid transport plays an important role in the nutrient supply for the oocyte. In male gametes, lipids are used as energy substrates in spermatozoa but nothing is yet known about their origin and metabolism throughout spermatogenesis. The lipid profiles analyzed from different stages of male germ cell development in the marine annelid Nereis virens were found to undergo a dramatic change from primary triacylglycerides at the beginning of germ cell development to cholesterol and phospholipids at the end of development as demonstrated by HPLC with evaporative light scattering detection and mass spectrometry. The uptake of a large discoidal lipoprotein into the developing germ cells could be demonstrated by fluorescence labeling and electron microscopic techniques as well as by the presence of a lipoprotein receptor in the germ cells, thus establishing its role in lipid supply. The incorporated lipoprotein discs were found to be stored as intact complexes indicating that they are not readily degraded upon endocytotic uptake. The change in lipid composition during germ cell development reflects their metabolic activity, especially in spermatogonia. The high concentration of lipids maintained by spermatogonia during the early phase of gametogenesis seems to be required for the later rapid processes of meiosis and spermatocyte differentiation. At times when peak demand of lipids arises for membrane synthesis and increased metabolism, this may be met more efficiently by a rapid on-site mobilization of lipids instead of an external supply.

 

2.128           Functional roles of VASP phosphorylation in the regulation of chemotaxis and osmotic stress response

Lin, W-H., Nelson, S.E., Hollingsworth, R.J. and Chung, C.Y.

Cytoskeleton, 67, 259-271 (2010)

 

Vasodilator-stimulated phosphoprotein (VASP) plays crucial roles in controlling F-actin-driven processes and growing evidence indicates that VASP function is modulated by phosphorylation at multiple sites. However, the complexity of mammalian system prevents the clear understanding of the role of VASP phosphorylation. In this study, we took advantage of Dictyostelium which possesses only one member of the Ena/VASP family to investigate the functional roles of VASP phosphorylation. Our results demonstrated that hyperosmotic stress and cAMP stimulation cause VASP phosphorylation. VASP phosphorylation plays a negative role for the early steps of filopodia/microspikes formation. VASP phosphorylation appears to modulate VASP localization at the membrane cortex and its interactions with WASP and WIPa. Analysis of chemotaxis of cells expressing VASP mutants showed that VASP phosphorylation is required for the establishment of cell polarity under a cAMP gradient.

 

2.129           Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 1. Pigment Stability and Light-induced Modifications of Lipoproteins

Dandler, J., Wilhelm, B. and Scheer, H.

Photochem. Photobiol., 86, 331-341 (2010)

 

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR′), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR′ is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.

 

2.130           Effect of changing the amount and type of fat and carbohydrate on insulin sensitivity and cardiovascular risk: the RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) trial

Jebb, S.A., Lovegrove, J.A., Griffin, B.A., Frost, G.S., Moore, C.S., Chatfield, M.D., Bluck, L.J., Williams, C.M. and Sanders, T.A.B.

Am. J. Clin. Nutr., 92, 748-758 (2010)

 

Background: Insulin sensitivity (Si) is improved by weight lossand exercise, but the effects of the replacement of saturatedfatty acids (SFAs) with monounsaturated fatty acids (MUFAs)or carbohydrates of high glycemic index (HGI) or low glycemicindex (LGI) are uncertain.

Objective: We conducted a dietary intervention trial to studythese effects in participants at risk of developing metabolicsyndrome.

Design: We conducted a 5-center, parallel design, randomizedcontrolled trial [RISCK (Reading, Imperial, Surrey, Cambridge,and Kings)]. The primary and secondary outcomes were changesin Si (measured by using an intravenous glucose tolerance test)and cardiovascular risk factors. Measurements were made after4 wk of a high-SFA and HGI (HS/HGI) diet and after a 24-wk interventionwith HS/HGI (reference), high-MUFA and HGI (HM/HGI), HM andLGI (HM/LGI), low-fat and HGI (LF/HGI), and LF and LGI (LF/LGI)diets.

Results: We analyzed data for 548 of 720 participants who were randomly assigned to treatment. The median Si was 2.7 x 10^–4 mL · µU^–1 · min^–1 (interquartile range: 2.0, 4.2 x 10^–4 mL · µU^–1 · min^–1), and unadjusted mean percentage changes (95% CIs) after 24 wk treatment (P = 0.13) were as follows: for the HS/HGIgroup, –4% (–12.7%, 5.3%); for the HM/HGI group,2.1% (–5.8%, 10.7%); for the HM/LGI group, –3.5%(–10.6%, 4.3%); for the LF/HGI group, –8.6% (–15.4%,–1.1%); and for the LF/LGI group, 9.9% (2.4%, 18.0%).Total cholesterol (TC), LDL cholesterol, and apolipoproteinB concentrations decreased with SFA reduction. Decreases inTC and LDL-cholesterol concentrations were greater with LGI.Fat reduction lowered HDL cholesterol and apolipoprotein A1and B concentrations.

Conclusions: This study did not support the hypothesis thatisoenergetic replacement of SFAs with MUFAs or carbohydrateshas a favorable effect on Si. Lowering GI enhanced reductionsin TC and LDL-cholesterol concentrations in subjects, with tentativeevidence of improvements in Si in the LF-treatment group. Thistrial was registered at clinicaltrials.gov as ISRCTN29111298.

 

2.131           Effects of antioxidants on postprandial oxidative stress and endothelial dysfunction in subjects with impaired glucose tolerance and Type 2 diabetes

Neri, S., Calvagno, S., Mauceri, B., Misseri, M., Tsami, A., Vecchio, C., Mastrosimone, G., Di Pino, A., Maiorca, D., Judica, A., Romano, G., Rizzotto, A. and Signorelli, S.S.

Eur. J. Nutr., 49, 409-416 (2010)

 

Aim  

To compare changes in the oxidation–reduction balance and endothelial function before and after meal in patients with type 2 diabetes or impaired glucose tolerance and determine the effects of standard antioxidant supplementation.

Methods  

Forty diabetics and 40 subjects with impaired glucose tolerance were compared with a control group. We assessed before and after a test meal (homogenized milkshake containing 80 g of saturated fat, amounting to 1,480 kcal), some reactive oxygen species, inflammation markers and flow-mediated vascular dilatation. These parameters were then reassessed after standard antioxidant treatment.

Results  

After the meal, diabetics, subjects with impaired glucose tolerance and controls had higher levels of oxidant compounds compared to fasting levels. In subjects with diabetes and impaired glucose tolerance (IGT), Vascular Adhesion Molecule-1 and CRP were higher after the meal—diabetic subjects exhibited lower fasting flow-mediated dilatation, which deteriorated significantly after the meal. Antioxidant administration significantly improved the parameters investigated in all subjects.

Conclusions  

In diabetic subjects, altered glycaemia and lipaemia are closely correlated with markers of systemic oxidative stress. Our results show that the abnormal changes in oxidative-reductive balance parameters are paralleled by similar changes in markers of endothelial dysfunction and inflammation at 4 h after ingestion of a fatty meal. Supplementation with a pool of antioxidants can reduce oxidative stress and inflammation in healthy subjects and, more importantly, in IGT patients. This previous aspect suggests that the timing of antioxidant supplementation has an important role in endothelium protection in healthy and pre-diabetic subjects, and along with prompt antioxidant treatment before irreversible endothelial damage has occurred, may have an important protective role in subjects with IGT—patients who require administration of adequate dietary antioxidants.

 

2.132           Role of the Highly Conserved Middle Region of Prion Protein (PrP) in PrP−Lipid Interaction

Wang, F., Yin, S., Wang, X., Zha, L., Sy, M-S. and Ma, J.

Biochemistry, 49, 8169-8176 (2010)

 

Converting normal prion protein (PrP^C) to the pathogenic PrP^Sc isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid−rPrP interaction in PrP conversion. To understand the mechanism of lipid−rPrP interaction, we analyzed the ability of various rPrP mutants to bind anionic lipids and to gain lipid-induced proteinase K (PK) resistance. We found that the N-terminal positively charged region contributes to electrostatic rPrP−lipid binding but does not affect lipid-induced PK resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP−lipid interaction and abolished the lipid-induced C-terminal PK resistance. The rPrP mutant without positive charges in the middle region reduced the amount of the lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain-localized 129 polymorphism altered the strength of hydrophobic rPrP−lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipids is essential for the formation of the PK-resistant conformation. Moreover, the influence of disease-associated PrP mutations and the 129 polymorphism on PrP−lipid interaction supports the relevance of PrP−lipid interaction to the pathogenesis of prion disease.

 

2.133           Oral Treatment with the d-Enantiomeric Peptide D3 Improves the Pathology and Behavior of Alzheimer’s Disease Transgenic Mice

Funke, S.A., van Groen, T., Kadish, I., Bartnik, D., Nage-Steger, L., Brener, O., Sehl, T., Batra-Safferling, R., Moriscot, C., Schoehn, G., Horn, A.H.C., Müller-Schiffmann, A., Korth, C., Sticht, H. and Willbold, D.

ACS Chem. Neurosci., 1(9), 639-648 (2010)

 

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Not only Aβ fibrils but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aβ targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

 

2.134           Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Eto, K., Noda, Y., Horikawa, S., Uchida, S. and Sasaki, S.

1. Biol. Chem., 285(52), 40777-40784 (2010)

 

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.

 

2.135           The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout

Prindiville, J.S., Mennigen, J.A., Zamora, J.M., Moon, T.W. and Weber, J-M.

Tox. Appl. Pharmacol., 251, 201-208 (2011)

 

Gemfibrozil (GEM) is a fibrate drug consistently found in effluents from sewage treatment plants. This study characterizes the pharmacological effects of GEM on the plasma lipoproteins of rainbow trout (Oncorhynchus mykiss). Our goals were to quantify the impact of the drug on: 1) lipid constituents of lipoproteins (phospholipids (PL), triacylglycerol (TAG), and cholesterol), 2) lipoprotein classes (high, low and very low density lipoproteins), and 3) fatty acid composition of lipoproteins. Potential mechanisms of GEM action were investigated by measuring lipoprotein lipase activity (LPL) and the hepatic gene expression of LPL and of the peroxisome proliferator-activated receptor (PPAR) α, β, and γ isoforms. GEM treatment resulted in decreased plasma lipoprotein levels (− 29%) and a reduced size of all lipoprotein classes (lower PL:TAG ratios). However, the increase in HDL-cholesterol elicited by GEM in humans failed to be observed in trout. Therefore, HDL-cholesterol cannot be used to assess the impact of the drug on fish. GEM also modified lipoprotein composition by reducing the abundance of long-chain n−3 fatty acids, thereby potentially reducing the nutritional quality of exposed fish. The relative gene expression of LPL was increased, but the activity of the enzyme was not, and we found no evidence for the activation of PPAR pathways. The depressing effects of GEM on fish lipoproteins demonstrated here may be a concern in view of the widespread presence of fibrates in aquatic environments. Work is needed to test whether exposure to environmental concentrations of these drugs jeopardizes the capacity of fish for reproduction, temperature acclimation or migratory behaviors.

 

2.136           Small dense LDL particles - a predictor of coronary artery disease evaluated by invasive and CT-based techniques: a case-control study

Toft-Petersen, A.P., Tilsted, H.H., aarøe, J., Rasmussen, K., Christensen, T., Griffin, B.A., Aardestrup, I.V., Andreasen, A. and Schmidt, E.B.

Lipids in Health and Disease, 10, 21-27 (2011)

 

Background

Coronary angiography is the current standard method to evaluate coronary atherosclerosis in patients with suspected angina pectoris, but non-invasive CT scanning of the coronaries are increasingly used for the same purpose.

Low-density lipoprotein (LDL) cholesterol and other lipid and lipoprotein variables are major risk factors for coronary artery disease. Small dense LDL particles may be of particular importance, but clinical studies evaluating their predictive value for coronary atherosclerosis are few.

Methods

We performed a study of 194 consecutive patients with chest pain, a priori considered of low to intermediate risk for significant coronary stenosis (>50% lumen obstruction) who were referred for elective coronary angiography. Plasma lipids and lipoproteins were measured including the subtype pattern of LDL particles, and all patients were examined by coronary CT scanning before coronary angiography.

Results

The proportion of small dense LDL was a strong univariate predictor of significant coronary artery stenosis evaluated by both methods. After adjustment for age, gender, smoking, and waist circumference only results obtained by traditional coronary angiography remained statistically significant.

Conclusion

Small dense LDL particles may add to risk stratification of patients with suspected angina pectoris.

 

2.137           LIPOPROTEIN SECRETION PROFILES AND VLDL PRODUCTION IN HEPATOCYTE CELL LINES

Jammart, B., Zoulin, F.and Durantel, D.

1. Hepatol., 54, S318 (2011)

 

Background and Aims: Hepatitis C virus (HCV) is highly associated to apolipoprotein-B containing lipoproteins (LDL and VLDL) in infected patient sera, as most viral RNA is co-immunoprecipitated

with anti-ApoB antibodies, but this association is barely seen in vitro (e.g. Huh7.5 cells infected with the HCV JFH-1 strain). Recent data suggested that these cells may be deficient for mature VLDL

production. Thus, our aim was to: i. characterize lipoprotein secretion in Huh7.5 cells; ii. compare this secretion to natural VLDL production by primary human hepatocytes (PHH); iii. find other hepatocyte cell lines competent for VLDL production.

Methods: Cell culture supernatants were harvested, concentrated using an Amicon centrifugal filter unit with a cut-off of 100 kDa (Millipore™) and ultracentrifuged over an iodixanolsucrose gradient. Density distributions of apolipoproteins were determined using ELISA or western blot. The production of mature

VLDL was further assessed by co-immunoprecipitation of different apolipoproteins.

Results: We found that Huh7.5 cells secrete a large amount of ApoB as compared to PHH. However, ApoB was detected at a density corresponding to LDL or IDL (sup. than 1.01 g/mL), but not VLDL, and was not associated with ApoE, as neither co-segregation nor co-immunoprecipitation were observed. Importantly, HCV infection increased ApoB secretion but did not affect the density of secreted particles. In contrast, secretion of ApoB/ApoE-containing lipoproteins with a density and composition comparable to that of PHH was observed in differentiated HepaRG cells, although the amount of secreted particles was lower. Finally, the hepatoblastoma cell line HepG2 was also able to secrete very-low-density particles

containing ApoB and ApoE upon treatment with oleic acid (to stimulate lipoprotein production) and MEK/ERK inhibitors (to reduce the over-activation of MEK1 kinase in these cells).

Conclusion: We have characterized lipoprotein secretion profiles in 3 different cell lines (Huh7.5, HepG2 and HepaRG) as well as in PHH. Huh7.5 cell line, which is commonly used to study HCV replication, does not seem to produce mature VLDL and is therefore a poor model to study HCV particle secretion and its association to lipoproteins as observed in vivo. The other hepatocyte cell lines may

therefore be more relevant study models of HCV morphogenesis.

 

2.138           The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles

Alkhatatbeh, M.J., Mhaidat, N.M., Enjeti, A.K., Lincz, L.F. and Thorne, R.F.

1. Thrombosis and Haemostasis, 9, 844-851 (2011)

 

Background:CD36 is a widely expressed cell surface receptor that binds  lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell-free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. Objective:To determine the  nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). Methods:Lipoproteins were fractionated by density gradient  centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight-488 anti-CD36 conjugate in combination with antibodies against different cellular markers. Results:Cell-free plasma CD36 was not  observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. Conclusions:sCD36  is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.

 

2.139           Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells

Detzer, A., Engel, C., Wûnsche, W. and Sczakiel, G.

Nucelic Acids Res., 39(7), 2727-2741 (2011)

 

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO₂), cycloheximide (CHX) and Lipofectamine^TM 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi.

Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO₂. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

 

2.140           Forming giant vesicles with controlled membrane composition, asymmetry, and contents

Richmond, D.L., Schmidt, E.M., Martens, S., Stachowiak, J.C., Liska, N. and Fletcher, D.A.

PNAS, 108(23), 9431-9436 (2011)

 

Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.

 

2.141           Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Reyes, A. et al

Nucleic Acid Res., 39(12), 5098-5108 (2011)

 

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

 

2.142           Macromolecular organization of ATP synthase and complex I in whole mitochondria

Davies, K.M., Strauss, M., Daum, B., Kief, J.H., Osiewacz, H.D., Rycovska, A., Zickermann, V and Kühlbrandt, W.

PNAS, 108(34), 14121-14126 (2011)

 

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F₁ heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

 

2.143           Spongiform Encephalopathy in Transgenic Mice Expressing a Point Mutation in the β2–α2 Loop of the Prion Protein

Sigurdson, C., Joshi-Barr, S., Bett, C., Winson, O., Manco, G., Schwarz, P., Rülicke, T., Nilsson, K.P.R., Margalith, I., Raeber, A., Peretz, D., Hornemann, S., Wüthrick, K. and Aguzzi, A.

1. Neurosci., 31(39), 13840-13847 (2011)

 

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrPC, into a β-sheet-rich, aggregated isoform, PrPSc. We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2–α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2–α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrPD167S developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2–α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

 

2.144           Fish Oil Supplementation During Late Pregnancy Does Not Influence Plasma Lipids or Lipoprotein Levels in Young Adult Offspring

Rytter, D., Schmidt, E.B., Bech, B.H., Christensen, J.H., henriksen, T.B. and Olsen, S.F.

Lipids, 46, 1091-1099 (2011)

 

Nutritional influences on cardiovascular disease operate throughout life. Studies in both experimental animals and humans have suggested that changes in the peri- and early post-natal nutrition can affect the development of the various components of the metabolic syndrome in adult life. This has lead to the hypothesis that n-3 fatty acid supplementation in pregnancy may have a beneficial effect on lipid profile in the offspring. The aim of the present study was to investigate the effect of supplementation with n-3 fatty acids during the third trimester of pregnancy on lipids and lipoproteins in the 19-year-old offspring. The study was based on the follow-up of a randomized controlled trial from 1990 where 533 pregnant women were randomized to fish oil (n = 266), olive oil (n = 136) or no oil (n = 131). In 2009, the offspring were invited to a physical examination including blood sampling. A total of 243 of the offspring participated. Lipid values did not differ between the fish oil and olive oil groups. The relative adjusted difference (95% confidence intervals) in lipid concentrations was −3% (−11; 7) for LDL cholesterol, 3% (−3; 10) for HDL cholesterol, −1% (−6; 5) for total cholesterol,−4% (−16; 10) for TAG concentrations, 2%(−2; 7) for apolipoprotein A1, −1% (−9; 7) for apolipoprotein B and 3% (−7; 15) in relative abundance of small dense LDL. In conclusion, there was no effect of fish oil supplementation during the third trimester of pregnancy on offspring plasma lipids and lipoproteins in adolescence.

 

2.145           An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Damas, J.E. and Cake, M.H.

Am. J. Physiol. Lung Cell. Mol. Physiol., 301(6), L966-L974 (2011)

 

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A₂ (PLA₂)-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA₂-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA₂. Furthermore, specific inhibitors of PLA₂ such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA₂-like activity may be an intrinsic attribute of albumin.

 

2.146           BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol

Geiger, R., Andritschke, D., Friebe, S., Herzog, F., Luisoni, S., heger, T. and Helenius, A.

Nature Cell Biol., 13(11), 1305-1314 (2011)

 

Non-enveloped viruses such as SV40 are transported from the extracellular space into the host cell nucleus through a pathway involving endocytosis, trafficking to the endoplasmic reticulum (ER) lumen, transport across the ER membrane to the cytoplasm, and subsequent nuclear import. Helenius and colleagues provide insight into how SV40 escapes from the ER by showing that viral proteins interact with components of the host ER-associated degradation machinery (ERAD). These interactions are crucial for translocation of SV40 into the cytoplasm and infectivity.

 

2.147           Does lycopene offer human LDL any protection against myeloperoxidase activity?

Chew, P.Y., Riley, L., Graham, D.L., Rahman, K. and Lowe, G.M.

Mol. Cell. Biochem., 361(1-2), 181-187 (2012)

 

Lycopene is a lipophilic antioxidant that is largely transported in human blood by Low Density Lipoproteins (LDL). One of the early events in the aetiology of atherosclerosis is thought to be the oxidation of LDL. Myeloperoxidase an enzyme secreted by neutrophils and macrophages is thought to oxidise human LDL particles. In this study, isolated human LDL was challenged with myeloperoxidase or copper, and the LDL was screened for lipoperoxidation and oxidation of apolipoprotein B100, depletion of lycopene and oxidation of cholesterol. Myeloperoxidase induced oxidation of LDL through direct interaction with apolipoprotein B100. No lipoperoxidation was observed following myeloperoxidase treatment; however, 7-ketocholesterol was detected indicating the products of myeloperoxidase interact with the surface of the LDL particles. Lycopene does react with the products of myeloperoxidase in solvent, but played no role in protecting against enzyme derived oxidation of human LDL.

 

2.148           Shedding of syndecan-1 from human hepatocytes alters very low density lipoprotein clearance

Deng, Y., Foley, E.M., Gonzales, J.C., Gordts, P.L., Li, Y. and Esko, J.D.

Hepatology, 55(1), 277-286 (2012)

 

We recently showed that the heparan sulfate proteoglycan syndecan-1 mediates hepatic clearance of triglyceride-rich lipoproteins in mice based on systemic deletion of syndecan-1 and hepatocyte-specific inactivation of sulfotransferases involved in heparan sulfate biosynthesis. Here, we show that syndecan-1 expressed on primary human hepatocytes and Hep3B human hepatoma cells can mediate binding and uptake of very low density lipoprotein (VLDL). Syndecan-1 also undergoes spontaneous shedding from primary human and murine hepatocytes and Hep3B cells. In human cells, phorbol myristic acid induces syndecan-1 shedding, resulting in accumulation of syndecan-1 ectodomains in the medium. Shedding occurs through a protein kinase C–dependent activation of ADAM17 (a disintegrin and metalloproteinase 17). Phorbol myristic acid stimulation significantly decreases DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)-VLDL binding to cells, and shed syndecan-1 ectodomains bind to VLDL. Although mouse hepatocytes appear resistant to induced shedding in vitro, injection of lipopolysaccharide into mice results in loss of hepatic syndecan-1, accumulation of ectodomains in the plasma, impaired VLDL catabolism, and hypertriglyceridemia. Conclusion: These findings suggest that syndecan-1 mediates hepatic VLDL turnover in humans as well as in mice and that shedding might contribute to hypertriglyceridemia in patients with sepsis.

 

2.149           Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria

Sharma, N.K., Reyes, A., Green, P., Caron, M.J., Bonini, M.G., Gordon, D.M., Holt, I.J. and Hertzog Santos, J.

Nucleic Acids Res., 40(2), 712-725 (2012)

 

Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

 

2.150           Large Aggregates Are the Major Soluble Aβ Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

Sehlin, D., Englund, H., Simu, B., Karlsson, M., Ingelsson, M., Nikolajeff, f., Lannfelt, L. and Pettersson, F.E.

PloS One, 7(2), e32014 (2012)

 

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.

 

2.151           The small G protein Arl1 directs the trans-Golgi–specific targeting of the Arf1 exchange factors BIG1 and BIG2

Christis, C. and Munro, S.

1. Cell. Biol., 196(3), 327-335 (2012)

 

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi–specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

 

2.152           Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

Huang, W., Choi, W., Hu, W., Mi, N., Guo, Q., Ma, M., Liu, M., Tian, Y., Lu, P., Wang, F-L., Deng, H., Liu, L., Gao, N., Yu, L. and Shi, Y.

Cell Res., 22, 473-489 (2012)

 

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

 

2.153           Density Gradient Multilayer Polymerization for Creating Complex Tissue

Karpiak, J.V., Ner, Y. and Almutairi, A.

Adv. Mater., 24(11), 1466-1470 (2012)

 

An adaptable density gradient multilayer polymerization (DGMP) method facilitates simple fabrication of complex multicompartment scaffolds with structurally continuous interfaces. Solvent density liquid-liquid phase segregation compartmentalizes varied mechanical and chemical cues independently. Bulk photopolymerization produces stratified three-dimensional and two-dimensional matrices. Cells attach to patterned adhesion peptides on biomimetic 2D substrates.

 

2.154           Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions

Butenhoff, J.L., Pieterman, E., Ehresman, D.J., Gorman, G.S., Olsen, G.W., Chang, S-C. and Princen, H.M.G.

Toxicology Letters, 210, 360-365 (2012)

 

Some cross-sectional epidemiological studies have reported positive associations of serum concentrations of non-high density lipoprotein cholesterol with serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). However, the strength of the reported associations is inconsistent for exposure–response across three orders of magnitude of serum PFOS and/or PFOA concentrations. These positive associations are unexpected based on toxicological/mechanistic studies, suggesting that the associations may have a biological, rather than a causal, basis. This study tested the hypothesis that PFOS and PFOA distribute into serum lipoprotein fractions such that increases in serum lipoproteins would result in corresponding increases in serum concentrations of PFOS and PFOA. Based on observed binding of PFOS and PFOA to isolated β-lipoproteins in physiological saline (96% and 40% bound, respectively) in preliminary experiments using ultrafiltration and LC–MS/MS methods, binding to human donor plasma lipoprotein fractions was investigated by two density gradient methods. The majority of PFOS and PFOA recovered masses were found in lipoprotein-depleted plasma. Plasma density gradient fractionation data suggested that maximally 9% of PFOS distributes to lipoprotein-containing fractions, yet only 1% or less of PFOA is so distributed. These data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose–response associations reported in cross-sectional epidemiological studies.

 

2.155           Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

Karatekin, E. and Ropthman, J.E.

Nature Protocols, 7(5), 903-920 (2012)

 

Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE–reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6–8 d to complete. Analysis can take up to 2 weeks.

 

2.156           P52—Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions over a wide range of concentrations

Butenhoff, J.L., Pieterman, E.J., Ehresman, D.J., Olsen, G.W., Chang, S-C. and Princen, H.M.G.

Reprod. Toxicol., 33, 1-29, abstract P-52 (2012)

 

Certain observational epidemiological studies have been characterized as finding modest positive associations between serum concentrations of non-high density lipoprotein cholesterol (non-HDL-C) and serum concentrations of PFOS and/or PFOA. However, collectively, these primarily cross-sectional investigations of occupational, community-exposed, and general populations are remarkably inconsistent for any dose–response relationship across a range of PFOS and/or PFOA concentrations. These PFOS and PFOA concentrations span three orders of magnitude between the least and highest exposed populations. Contrary to the epidemiological positive associations, toxicological studies of PFOS and PFOA in laboratory animals, including cynomologus monkeys, have observed either no change in serum lipids or decreases in serum cholesterol and/or triglycerides. These conflicting observations suggest that the reported epidemiologic associations of serum PFOS and PFOA with serum cholesterol may have biological, but not causal, significance. A possible non-causal hypothesis for the reported epidemiological associations between serum PFOS and/or PFOA and serum cholesterol is that PFOS and/or PFOA have affinity for and distribute into serum lipoprotein fractions. Thus, it can be postulated that, as serum lipoprotein concentration increases, distribution of PFOS and PFOA into serum lipoprotein fractions and total serum PFOS and/or PFOA concentrations would increase correspondingly. The study reported herein was undertaken to test the latter hypothesis through investigation of the effect of plasma concentration of PFOS and PFOA on the proportion of plasma PFOS and PFOA bound to the human plasma lipoprotein fractions VLDL, LDL, and HDL at background general population serum PFOS and PFOA concentrations and in serum spiked with either approximately 0.19 or 19 μM PFOS or PFOA. Plasma from a senior investigator was obtained and used to represent general population background concentrations of PFOS and PFOA, as well as to prepare approximately 0.19 and 19 μM concentrations of each fluorochemical. All experiments were performed in triplicate. Fractionation of 300 μL plasma into 25 fractions was accomplished by density gradient ultracentrifugation using both Redgrave and iodixanol density gradients. For each density gradient, fractions were pooled into very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and lipoprotein depleted plasma (LPDP) fractions as well as intermediate fractions based on determination of cholesterol concentrations in each of 25 fractions. Both density gradients were adjusted to give clean separations between the lipoprotein fractions and between HDL and albumin contained in LPDP. PFOS and PFOA concentrations in pooled fractions were determined by LC–MS/MS with lower limits of quantitation (LLOQ) ranging from 10 to 25 pg/mL. Total recovered mass of PFOS and PFOA based on duplicate determinations of initial concentrations ranged from 71 to 84% and 95 to 109% for PFOS and from 82 to 102% and 96 to 110% for PFOA for Redgrave and iodixanol density gradients, respectively. The majority of PFOS and PFOA recovered mass was found in LPDP (52–60% and 87–104% of PFOS mass and 81–98% and 94–109% of PFOA mass for Redgrave and iodixanol density gradients, respectively). Percent of mass recovered from fractions 1 to 19 (lipoprotein-containing fractions) ranged from 17 to 24% and <5 to 9% for PFOS and from <4 to <8% and <1 to <4% for PFOA for the Redgrave and iodixanol density gradients, respectively. PFOS concentrations in LDL and HDL were clearly higher than in VLDL and intermediate pooled lipoprotein fractions. For both PFOS and PFOA, there was minimal difference between concentrations tested and the resulting proportion distributed to the various pooled fractions. The results using the iodixanol method may be more representative physiologically, as iodixanol is a water soluble, non-ionic fluid. The iodixanol data suggest that maximally 9% of PFOS may be distributed to lipoprotein-containing fractions in plasma, yet only 1% or less of PFOA is so distributed. Taken together, these data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose–response associations reported in observational epidemiological studies.

 

2.157           Identification and Characterization of an Aβ Oligomer Precipitating Peptide That May Be Useful to Explore Gene Therapeutic Approaches to Alzheimer Disease

Funke, S.A:, Liu, H., Sehl, T., Bartnik, D., Brener, O., Nagel-Steger, L., Wiesehan, K. and Wilbold, D.

Rejuvenation Res., 15(2), 144-147 (2012)

 

A key feature of Alzheimer disease (AD) is the pathologic self-association of the amyloid-β (Aβ) peptide, leading to the formation of diffusible toxic Aβ oligomers and extracellular amyloid plaques. Next to extracellular Aβ, intraneuronal Aβ has important pathological functions in AD. Agents that specifically interfere with the oligomerization processes either outside or inside of neurons are highly desired for the elucidation of the pathologic mechanisms of AD and might even pave the way for new AD gene therapeutic approaches. Here, we characterize the Aβ binding peptide L3 and its influence on Aβ oligomerization in vitro. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added Aβ in neuroblastoma cells.

 

2.158           A Dual Role for UVRAG in Maintaining Chromosomal Stability Independent of Autophagy

Zhao, Z., Oh, S., Li, D., Ni, D., Pirooz, S.D., Lee, J-H., yang, S., Lee, J-Y., Ghozalli, I., Costanzo, V., Stark, J.M. and Liang, C.

Developmental Cell, 22(5), 1-16 (2012)

 

Autophagy defects have recently been associated with chromosomal instability, a hallmark of human cancer. However, the functional specificity and mechanism of action of autophagy-related factors in genome stability remain elusive. Here we report that UVRAG, an autophagic tumor suppressor, plays a dual role in chromosomal stability, surprisingly independent of autophagy. We establish that UVRAG promotes DNA double-strand-break repair by directly binding and activating DNA-PK in nonhomologous end joining. Disruption of UVRAG increases genetic instability and sensitivity of cells to irradiation. Furthermore, UVRAG was also found to be localized at centrosomes and physically associated with CEP63, an integral component of centrosomes. Disruption of the association of UVRAG with centrosomes causes centrosome instability and aneuploidy. UVRAG thus represents an autophagy-related molecular factor that also has a convergent role in patrolling both the structural integrity and proper segregation of chromosomes, which may confer autophagy-independent tumor suppressor activity.

 

2.159           Differential Effects of Grape (Vitis vinifera) Skin Polyphenolics on Human Platelet Aggregation and Low-Density Lipoprotein Oxidation

Shanmuganayagam, D., Beahm, M.R., Kuhns, M.A., Krueger, C.G., Reed, J.D. and Folts, J.D.

1. Agric. Food Chem., 60(23), 5787-5794 (2012)

 

Antioxidant and antiplatelet properties of grape products are thought to be responsible for observed antiatherosclerotic effects. Diverse classes of phenolics are derived from the seed and skin (GSK) of grapes. The relative contributions of the classes of phenolics to observed properties of grape products are unknown. In this paper, GSK fractions were used to examine effects on platelet aggregation, low-density lipoprotein (LDL) oxidation in vitro, and relative binding of phenolics to LDL. GSK was separated into six fractions (fractions 1–6), and primary phenolics were characterized using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fractions 4, 5, and 6, enriched in polygalloyl polyflavan-3-ols (PGPFs) with 3–6, 4–8, and 6–15 degrees of polymerization, respectively, inhibited platelet aggregation. Fractions 1–3, containing various amounts of oligosaccharides, hydroxycinnamic acids, anthocyanins, flavanols, and low molecular weight PGPFs, significantly increased platelet aggregation. Fractions 4–6 were most effective in binding LDL and inhibiting LDL oxidation. Fractions 5 and 6 exhibited the greatest inhibition of platelet aggregation and LDL oxidation, suggesting that polymeric PGPFs are responsible for the beneficial effects of grape products. Conversely, phenolics in fractions 1–3 may reduce the net biological potency of the grape products and have undesirable effects on cardiovascular disease risk factors.

 

2.160           , 58-63 (2012)small mitochondrial ribosomal subunit

He, J., Cooper, H.M., Reyes, A., Di Re, M., Kazak, L., Wood, S.R., Mao, C.C., Fearnley, I.M., Walker, J.E. and Holt, I.J.

Nucleic Acids Res., 40(13), 6097-6108 (2012)

 

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

 

2.161           Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

He, J., Cooper, H.M., Reyes, A., Di Re, M., Sembongi, H., Litwin, T.R., Gao, J., Neuman, K.C., Fearnley, I.M., Spinazzola, A., Walker, J.E. and Holt, I.J.

Nucleic Acids Res., 40(13), 6109-6121 (2012)

 

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

 

2.162           Benzophenones and xanthones from Garcinia cantleyana var. cantleyana and their inhibitory activities on human low-density lipoprotein oxidation and platelet aggregation

Jantan, I. and Saputri, C.

Phytochemistry, 80, 58-63 (2012)

 

Three benzophenones, 2,6,3′,5′-tetrahydroxybenzophenone (1), 3,4,5,3′,5′-pentahydroxybenzophenone (3) and 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone (4), as well as a xanthone, 1,3,6-trihydroxy-5-methoxy-7-(3′-methyl-2′-oxo-but-3′-enyl)xanthone (9), were isolated from the twigs of Garcinia cantleyana var. cantleyana. Eight known compounds, 3,4,5,3′-tetrahydroxy benzophenone (2), 1,3,5-trihydroxyxanthone (5), 1,3,8-trihydroxyxanthone (6), 2,4,7-trihydroxyxanthone (7), 1,3,5,7-tetrahydroxyxanthone (8), quercetin, glutin-5-en-3β-ol and friedelin were also isolated. The structures of the compounds were elucidated by spectroscopic methods. The compounds were investigated for their ability to inhibit low-density lipoprotein (LDL) oxidation and platelet aggregation in human whole blood in vitro. Most of the compounds showed strong antioxidant activity with compound 8 showing the highest inhibition with an IC₅₀ value of 0.5 μM, comparable to that of probucol. Among the compounds tested, only compound 4 exhibited strong inhibitory activity against platelet aggregation induced by arachidonic acid (AA), adenosine diphosphate (ADP) and collagen. Compounds 3, 5 and 8 showed selective inhibitory activity on platelet aggregation induced by ADP.

 

2.163           Purified and synthetic Alzheimer’s amyloid beta (Aβ) prions

Stöhr, J., Watts, J.C., Mensinger, Z.L., Oehler, A., Grillo, S.K., DeArmond, S.J., Prusiner, S.B. and Giles, K.

PNAS, 109(27), 11025-11030 (2012)

 

The aggregation and deposition of amyloid-β (Aβ) peptides are believed to be central events in the pathogenesis of Alzheimer’s disease (AD). Inoculation of brain homogenates containing Aβ aggregates into susceptible transgenic mice accelerated Aβ deposition, suggesting that Aβ aggregates are capable of self-propagation and hence might be prions. Recently, we demonstrated that Aβ deposition can be monitored in live mice using bioluminescence imaging (BLI). Here, we use BLI to probe the ability of Aβ aggregates to self-propagate following inoculation into bigenic mice. We report compelling evidence that Aβ aggregates are prions by demonstrating widespread cerebral β-amyloidosis induced by inoculation of either purified Aβ aggregates derived from brain or aggregates composed of synthetic Aβ. Although synthetic Aβ aggregates were sufficient to induce Aβ deposition in vivo, they exhibited lower specific biological activity compared with brain-derived Aβ aggregates. Our results create an experimental paradigm that should lead to identification of self-propagating Aβ conformations, which could represent novel targets for interrupting the spread of Aβ deposition in AD patients.

 

2.164           Feedback Regulation of Transcriptional Termination by the Mammalian Circadian Clock PERIOD Complex

Padmanabhan, K. eet al

Science, 337, 599-602 (2012)

 

Eukaryotic circadian clocks are built on transcriptional feedback loops. In mammals, the PERIOD (PER) and CRYPTOCHROME (CRY) proteins accumulate, form a large nuclear complex (PER complex), and repress their own transcription. We found that mouse PER complexes included RNA helicases DDX5 and DHX9, active RNA polymerase II large subunit, Per and Cry pre-mRNAs, and SETX, a helicase that promotes transcriptional termination. During circadian negative feedback, RNA polymerase II accumulated near termination sites on Per and Cry genes but not on control genes. Recruitment of PER complexes to the elongating polymerase at Per and Cry termination sites inhibited SETX action, impeding RNA polymerase II release and thereby repressing transcriptional reinitiation. Circadian clock negative feedback thus includes direct control of transcriptional termination.

 

2.165           The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P2 synthesis

Ling, Y., Stefan, C.J., MacGurn, J.A., Audhya, A. and Emr, S.D.

EMBO J., 31(13), 2882-2894 (2012)

 

Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P₂, is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P₂ at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P₂ synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P₂ and serves as a negative regulator of PtdIns(4,5)P₂ synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling.

 

2.166           Lipoproteins in Drosophila melanogaster—Assembly, Function, and Influence on Tissue Lipid Composition

Palm, W., Sampaio, J.L., Brankatschk, M., Carvalho, M., Mahmoud, A., Shevchenko, A. and Eaton, S.

PloS One, 8(7), e1002828 (2012)

 

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo–synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization.

 

 

2.167           Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

Shutova, M., Yang, C., Vasiliev, J.M. and Svitkina, T.

PloS One, 7(7), e40814 (2012)

 

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

 

2.168           Structural and genetic basis for development of broadly neutralizing influenza antibodies

Lingwood, D., McTamney, P., Yassine, H.M., Whittle, J.R., Guo, X., Boyington, J.C., Wei, C-J. and Nabel, G.J.

Nature, 487, 566-570 (2012)

 

Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors^{1, 2}, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural ‘patterns’ that provide a substrate for further affinity maturation.

 

2.169           Small dense LDL: An emerging risk factor for cardiovascular disease

Hirayama, S. and Miida, T.

Clinica Chemica Acta, 414, 215-224 (2012)

 

Although low-density lipoprotein cholesterol (LDL-C) is a strong risk factor for coronary artery disease (CAD), LDL-C levels are not always elevated in CAD patients. LDL consists of several subclasses with distinct sizes, densities, and physicochemical compositions. Thus, LDL subclasses can be separated by various laboratory procedures. Among them, ultracentrifugation and electrophoresis have been used most frequently for determining LDL subclasses. Accumulating evidence has shown that a predominance of small dense LDL (sd-LDL) is closely associated with CAD. Moreover, sd-LDL-cholesterol (sd-LDL-C) concentrations are elevated in groups at a high risk for CAD, such as patients with type 2 diabetes and metabolic syndrome. Therefore, sd-LDL concentration is recognized as a surrogate marker for CAD. However, some studies failed to show therapeutic modulation of sd-LDL, likely because separating methods and sd-LDL particle definitions have not yet been standardized. Recently, a detergent-based homogenous assay for sd-LDL-C has been developed. This method does not require any pretreatment, and the measured values are highly reproducible with an automated analyzer. These features are suitable for large-scale clinical studies. This homogeneous assay is a useful tool for clarifying whether sd-LDL-C is a superior marker to LDL-C, and whether sd-LDL-C lipid-lowering therapies decrease the incidence of CAD.

 

2.170           Inhibitory Activities of Compounds from the Twigs of Garcinia hombroniana Pierre on Human Low-density Lipoprotein (LDL) Oxidation and Platelet Aggregation

Saputri, F.C. and Jantan, I.

Phytother. Res., 26(12), 1845-1850 (2012)

 

The methanol extract of the twigs of Garcinia hombroniana, which showed strong LDL antioxidation and antiplatelet aggregation activities, was subjected to column chromatography to obtain 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone, 1,7-dihydroxyxanthone and eight triterpenoids, garcihombronane B, D, E and F, friedelin, glutin-5-en-3β-ol, stigmasterol and lupeol. The structures of the compounds were elucidated by spectroscopic methods. The compounds were evaluated for their ability to inhibit copper-mediated LDL oxidation and arachidonic acid (AA)-, adenosine diphosphate (ADP)-, collagen-induced platelet aggregation in vitro. Among the compounds tested, 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone and 1,7-dihydroxyxanthone showed strong inhibitory activity on LDL oxidation with half-maximal inhibitory concentration (IC₅₀) values of 6.6 and 1.7 µm, respectively. 3,5,3′,5′-Tetrahydroxy-4-methoxybenzophenone exhibited strong activity on AA-, ADP- and collagen-induced platelet aggregation with IC₅₀ values of 53.6, 125.7 and 178.6 µm, respectively, while 1,7 dihydroxyxanthone showed significant and selective inhibitory activity against ADP-induced aggregation with IC₅₀ value of 5.7 µm. Of the triterpenoids tested, garcihombronane B showed moderate activity against LDL oxidation and garcihombronane D and F showed selective inhibition on ADP-induced platelet aggregation.

2.171           Why working with porcine circulating serum amyloid A is a pig of a job

Soler, L., Molenaar, A., Merola, N., Eckersall, P.D., Butierrez, A., Ceron, J.J.., Mulero, V. and Niewold, T.A:

J. Theoretical Biol., 317, 119-125 (2012)

 

Serum amyloid A (SAA) is a major acute phase protein in most species, and is widely employed as a health marker. Systemic SAA isoforms (SAA1, and SAA2) are apolipoproteins synthesized by the liver which associate with high density lipoproteins (HDL). Local SAA (SAA3) isoforms are synthesized in other tissues and are present in colostrums, mastitic milk and mammary dry secretions. Of systemic SAA the bulk is monomeric and bound to HDL, and a small proportion is found in serum in a multimeric form with a buried HDL binding site. In most species, systemic SAA could easily be studied by purifying it from serum of diseased individuals by hydrophobic interaction chromatography methods. For years, we were not able to isolate systemic pig SAA using the latter methods, and found that the bulk of pig SAA did not reside in the HDL-rich serum fractions but in the soluble protein fraction mainly as a multimeric protein.

Based on these surprising results, we analysed in silico the theoretical properties and predicted the secondary structure of pig SAA by using the published pig primary SAA amino acid sequence. Results of the analysis confirmed that systemic pig SAA had the highest homology with local SAA3 which in other species is the isoform associated with non-hepatic production in tissues such as mammary gland and intestinal epithelium. Furthermore, the primary sequence of the pig SAA N-terminal HDL binding site did differ considerably from SAA1/2. Secondary structure analysis of the predicted alpha–helical structure of this HDL binding site showed a considerable reduction in hydrophobicity compared to SAA1/2. Based on these results, it is argued that systemic acute phase SAA in the pig has the structural properties of locally produced SAA (SAA3). It is proposed that in pig SAA multimers the charged N-terminal sequence is buried, which would explain their different properties.

It is concluded that pig systemic SAA is unique compared to other species, which raises questions about the proposed importance of acute phase SAA in HDL metabolism during inflammation in this species.

 

2.172           Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird, N.L., York, J. and Nunberg, J.H.

J. Virol., 86(20), 11301-11310 (2012)

 

Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.

 

2.173           Alternative translation initiation augments the human mitochondrial proteome

Kazak, L., Reyes, A., Duncan, A.L., Rorbach, J., Wood, S.R., Brea-Calvo, g., Gammage, P.A., Robinson, A.J., Minczuk, M. and Holt, I.J.

Nucleic Acids Res., 41(4), 2354-2369 (2013)

 

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.

 

2.174           FisB mediates membrane fission during sporulation in Bacillus subtilis

Doan, T.., Coleman, J., Marquis, K.A. et al

Gens Dev., 27(3), 322-334 (2013)

 

How bacteria catalyze membrane fission during growth and differentiation is an outstanding question in prokaryotic cell biology. Here, we describe a protein (FisB, for fission protein B) that mediates membrane fission during the morphological process of spore formation in Bacillus subtilis. Sporulating cells divide asymmetrically, generating a large mother cell and smaller forespore. After division, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Membrane fission releases the forespore into the mother cell cytoplasm. Cells lacking FisB are severely and specifically impaired in the fission reaction. Moreover, GFP-FisB forms dynamic foci that become immobilized at the site of fission. Purified FisB catalyzes lipid mixing in vitro and is only required in one of the fusing membranes, suggesting that FisB–lipid interactions drive membrane remodeling. Consistent with this idea, the extracytoplasmic domain of FisB binds with remarkable specificity to cardiolipin, a lipid enriched in the engulfing membranes and regions of negative curvature. We propose that membrane topology at the final stage of engulfment and FisB–cardiolipin interactions ensure that the mother cell membranes are severed at the right time and place. The unique properties of FisB set it apart from the known fission machineries in eukaryotes, suggesting that it represents a new class of fission proteins.

 

2.175           Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains

Volmer, R., van der Ploeg, K. and Ron, D.

PNAS, 110(12), 4628-4633 (2013)

 

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1α and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.

 

2.176           Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Kosenko, T., Golder, M., Leblond, G., Weng, W. and Lagace, T.A.

1. Biol. Chem., 288(12), 8279-8288 (2013)

 

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K_D ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.

 

2.177           Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

Palm, W., Swierczynska, M.M., Kumari, V., Ehrhart-Bornstein, M., Bornstein, S.R. and Eaton, S.

PloS Biology, 11(3), e1001505 (2013)

 

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses

 

2.178           Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

Jammart, B., Michelet, M., Pecheur, E-I., parent, R., bartosch, B., Zoulim, F. and Durantel, D.

1. Virol., 87(9), 5065-5080 (2013)

 

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

 

2.179           Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

Bonander, N., Jamshad, M., Oberthür, D., Clare, M., Barwell, J., Hu, K., Farquhar, M.J., Stamataki, Z., Harris, H.J., Dierks, K., daffron, T.R., Betzel, C., McKeating, J.A. and Bill, R.M.

PloS One, 8(5), e64517 (2013)

 

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

 

2.180           A Cryptic Targeting Signal Creates a Mitochondrial FEN1 Isoform with Tailed R-Loop Binding Properties

Kazak, L., Reyes, A., He, J., Wood, S.R., Brea-Calvo, G., Holen, T.T. and Holt, I.J.

PloS One, 8(5), e62340 (2013)

 

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5′ RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

 

2.181           Separation of the principal HDL subclasses by iodixanol ultracentrifugation

Harman, N.L., Griffin, B.A. and Davies, I.G.

1. Lipid Res., 54, 2273-2281 (2013)

 

HDL subclasses detection, in cardiovascular risk, has been limited due to the time-consuming nature of current techniques. We have developed a time-saving and reliable separation of the principal HDL subclasses employing iodixanol density gradient ultracentrifugation (IxDGUC) combined with digital photography. HDL subclasses were separated in 2.5 h from prestained plasma on a three-step iodixanol gradient. HDL subclass profiles were generated by digital photography and gel scan software. Plasma samples (n = 46) were used to optimize the gradient for the resolution of HDL heterogeneity and to compare profiles generated by IxDGUC with gradient gel electrophoresis (GGE); further characterization from participants (n = 548) with a range of lipid profiles was also performed. HDL subclass profiles generated by IxDGUC were comparable to those separated by GGE as indicated by a significant association between areas under the curve for both HDL₂ and HDL₃ (HDL₂, r = 0.896, P < 0.01; HDL₃, r = 0.894, P < 0.01). The method was highly reproducible, with intra- and interassay coefficient of variation percentage < 5 for percentage area under the curve HDL₂ and HDL₃, and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses.

 

2.182           Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain

Vijayakumar, S., Peng, H. and Schwartz, G.J.

Am. J: Physiol. Renal Physiol., 305, F90-F99 (2013)

 

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for β-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH₂-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

 

2.183           Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein Receptor: Evidence for a Reciprocal Regulation

Tavori, H., Fan, D., Blakemore, J.L., Yancey, P.G., Ding, L., MacRae, F.-L. and Fazio, S.

Circulation, 127, 2403-2413 (2013)

 

Background—Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation.

Methods and Results—To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR^−/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR^+/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR^−/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression.

Conclusions—Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

 

2.184           Very low-density lipoprotein/lipo-viro particles reverse lipoprotein lipase-mediated inhibition of hepatitis C virus infection via apolipoprotein C-III

Sun, H-Y., Lin, C-C., Lee, J-C., Wang, S-W., Cheng, P-N., Wu, I-C., Chang, T-T., Lai, M-D., Shieh, D.B. and Young, K-C.

Gut, 62(8), 1193-1203 (2013)

 

Objective Circulating hepatitis C virus (HCV) virions are associated with triglyceride-rich lipoproteins, including very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), designated as lipo-viro-particles (LVPs). Previous studies showed that lipoprotein lipase (LPL), a key enzyme for hydrolysing the triglyceride in VLDL to finally become LDL, may suppress HCV infection. This investigation considers the regulation of LPL by lipoproteins and LVPs, and their roles in the LPL-mediated anti-HCV function.

Design The lipoproteins were fractionated from normolipidemic blood samples using iodixanol gradients. Subsequent immunoglobulin-affinity purification from the canonical VLDL and LDL yielded the corresponding VLDL-LVP and LDL-LVP. Apolipoprotein (apo) Cs, LPL activity and HCV infection were quantified.

Results A higher triglyceride/cholesterol ratio of LDL was found more in HCV-infected donors than in healthy volunteers, and the triglyceride/cholesterol ratio of LDL-LVP was much increased, suggesting that the LPL hydrolysis of triglyceride may be impaired. VLDL, VLDL-LVP, LDL-LVP, but not LDL, suppressed LPL lipolytic activity, which was restored by antibodies that recognised apoC-III/-IV and correlated with the steadily abundant apoC-III/-IV quantities in those particles. In a cell-based system, treatment with VLDL and LVPs reversed the LPL-mediated inhibition of HCV infection in apoC-III/-IV-dependent manners. A multivariate logistic regression revealed that plasma HCV viral loads correlated negatively with LPL lipolytic activity, but positively with the apoC-III content of VLDL. Additionally, apoC-III in VLDL was associated with a higher proportion of HCV-RNA than was IgG.

Conclusion This study reveals that LPL is an anti-HCV factor, and that apoC-III in VLDL and LVPs reduces the LPL-mediated inhibition of HCV infection.

 

 

2.185           Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Laferriere, F., Tixador, P., Moudjou, M., Chapuis, J., Sibille, P., Herzog, L., Reine, F., Jaumain, E., Laude, H., Rezaei, H. and Beringue, V.

PloS Pathogens, 9(10), e1003702 (2013)

 

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.

 

2.186           Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid

Lee, K-W., Okot-Kotber, C., LaComb, J.F. and Bogenhagen, D.F.

1. Biol. Chem., 288(43), 31386-31399 (2013)

 

We have identified RNMTL1, MRM1, and MRM2 (FtsJ2) as members of the RNA methyltransferase family that may be responsible for the three known 2′-O-ribose modifications of the 16 S rRNA core of the large mitochondrial ribosome subunit. These proteins are confined to foci located in the vicinity of mtDNA nucleoids. They show distinct patterns of association with mtDNA nucleoids and/or mitochondrial ribosomes in cell fractionation studies. We focused on the role of the least studied protein in this set, RNMTL1, to show that this protein interacts with the large ribosomal subunit as well as with a series of non-ribosomal proteins that may be involved in coupling of the rate of rRNA transcription and ribosome assembly in mitochondria. siRNA-directed silencing of RNMTL1 resulted in a significant inhibition of translation on mitochondrial ribosomes. Our results are consistent with a role for RNMTL1 in methylation of G¹³⁷⁰ of human 16 S rRNA.

 

2.187           Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

Daus, M.L., Wagenführ, K., Thomzig, A., Boerner, S., Hermann, P., Hermelink, A., Beekes, M. and Lasch, P.

1. Biol. Chem., 288(49), 35068-35080 (2013)

 

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.

 

2.188           Loss of Plasma Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) After Lipoprotein Apheresis

Tavori, H., Giunzioni, I., MacRae, F.L. and Fazio, S.

Circ. Res., 113, 1290-1295 (2013)

 

Rationale: Lipoprotein apheresis (LA) reduces low-density lipoprotein (LDL) levels in patients with severe familial hypercholesterolemia (FH). We have recently reported that >30% of plasma proprotein convertase subtilisin/kexin 9 (PCSK9) is bound to LDL, thus we predicted that LA would also reduce plasma PCSK9 levels by removing LDL.

Objective: Pre- and post-apheresis plasma from 6 patients with familial hypercholesterolemia on 3 consecutive treatment cycles was used to determine changes in PCSK9 levels.

Methods and Results: LA drastically reduced plasma LDL (by 77±4%). Concomitantly, PCSK9 levels fell by 52±5%, strongly correlating with the LDL drop (P=0.0322; r²=0.26), but not with decreases in triglyceride (49±13%) or high-density lipoprotein levels (18±2%). Levels of albumin, creatinine, and CK-MB did not show significant changes after LA. Similar to LDL, PCSK9 levels returned to pretreatment values between cycles (2-week intervals). Fractionation of pre- and post-apheresis plasma showed that 81±11% of LDL-bound PCSK9 and 48±14% of apolipoprotein B–free PCSK9 were removed. Separation of whole plasma, purified LDL, or the apolipoprotein B–free fraction through a scaled-down, experimental dextran sulfate cellulose beads column produced similar results.

Conclusions: Our results show, for the first time, that modulation of LDL levels by LA directly affects plasma PCSK9 levels, and suggest that PCSK9 reduction is an additional benefit of LA. Because the loss of PCSK9 could contribute to the LDL-lowering effect of LA, then (1) anti-PCSK9 therapies may reduce frequency of LA in patients currently approved for therapy, and (2) LA and anti-PCSK9 therapies may be used synergistically to reduce treatment burden.

 

2.189           PrimPol, an Archaic Primase/Polymerase Operating in Human Cells

Garcia-Gomez, S., Reyes, A., Martinez-Jimenez, M.I., Chocron, E.S., Mouron, S., Terrados, G., Powell, C., Salido, E., Mendez, J., Holt, I.J. and Blanco, L.

Molecular  Cell, 52, 541-553 (2013)

 

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

 

2.190           Interactome of Two Diverse RNA Granules Links mRNA Localization to Translational Repression in Neurons

Fritzsche, R. et al

Cell Reports, 5, 1749-1762 (2013)

 

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

 

2.191           Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

Hesse, D., Radloff, K., Jaschke, A., Lagerpusch, M., Chung, B., Tailleux, A., Staels, B. and Schürmann, A.

1. Lipid Res., 55, 41-52 (2014)

 

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.

 

2.192           The 51 kDa FADS3 is Secreted in the ECM of Hepatocytes and Blood in Rat

Blanchard, H., Boulier-Monthean, N., Legrand, P. and Pedrono, F.

1. Cell. Biochem., 115(1), 199-207 (2014)

 

The fatty acid desaturase (Fads) cluster is composed of three genes encoding for the Δ5- and Δ6-desaturases and FADS3. The two former proteins are involved in the fatty acid biosynthesis; the latter one shares a high sequence identity but has still no attributed function. In a previous work performed in rat, we described three isoforms of FADS3 expressed in a tissue-dependent manner. In the present study, we demonstrated a specific subcellular targeting depending on the isoform. In cultured hepatocytes, which mainly expressed the 51 kDa protein, FADS3 was unexpectedly present in the cytosolic fraction, but was also secreted in the extracellular matrix on fibronectin-containing fibers. The secretion pathway was investigated and we determined the presence of exosome-like vesicles on the FADS3-stained fibers. In parallel, FADS3 was detected in blood of hepatic vessel, and particularly in serum. In conclusion, this study demonstrated a very specific intra- and extracellular location of FADS3 in comparison with the Δ5- and Δ6-desaturases, suggesting a unique function for this putative desaturase, even if no activity has been yet identified neither in the extracellular matrix of hepatocytes nor in serum.

 

2.193           Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

Coleman, B.M., Harrison, C.F., Guo, B., Masters, C.L., Barnham, K.J., Lawson, V.A. and Hill, A.F.

1. Virol., 88(5), 2690-2703 (2014)

 

Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP^C, into the disease-associated isoform, PrP^Sc. Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP^C share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP^Sc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP^Sc, giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion.

 

2.194           Immobilization of Homogeneous Monomeric, Oligomeric and Fibrillar Aβ Species for Reliable SPR Measurements

Frenzel, D., Glück, J.M., Brener, O., Oesterhelt, F., Nagel-Steger, L. and Willbold, D.

PloS One, 9(3), e89490 (2014)

 

There is strong evidence that the amyloid-beta peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aβ conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aβ allows reliable SPR measurements and quality control of the immobilized Aβ aggregate species at any step throughout the experiment.

 

2.195           Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

Rajala, N., Gerhold, J.M., Martinsson, P., Klymov, A. and Spelbrink, J.N.

Nucleic Acids Res., 42(2), 952-967 (2014)

 

Mitochondrial DNA (mtDNA) is organized in discrete protein–DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.

 

2.196           Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids

Borgenhagen, D.F., martin, D.W. and Koller, A.

Cell Metabolism, 19, 618-629 (2014)

 

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.

 

2.197           Regulated Oligomerization Induces Uptake of a Membrane Protein into COPII Vesicles Independent of Its Cytosolic Tail

Springer, S., Malkus, P., Borchert, B., Wellbrock, U., Duden, R. and Schekman, R.

Traffic, 15, 531-545 (2014)

 

Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)-coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII-coat-forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that – owing to its FK506-binding protein domains – can be oligomerized in isolated membranes by addition of a small-molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization-dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.

 

 

 

2.198           The metabolic inter-relationships between changes in waist circumference, triglycerides, insulin sensitivity and small, dense low-density lipoprotein particles with acute weight loss in clinically obese children and adolescents

Hobkirk, J.P., King, R.E., davies, I., Harman, N., Gately, P., Pemberton, P., Smith, A., barth, J.H. and Carroll, S.

Pediatric Obesity, 9(3), 209-217 (2014)

 

Objective

Small, dense low-density lipoprotein (LDL) particles are highly atherogenic and strongly associated with obesity-related dyslipidemia. The metabolic inter-relationships between weight loss induced changes in waist circumference, triglycerides, insulin sensitivity and small-dense LDL particles in clinically obese children and adolescents have not been studied.

Methods

Seventy-five clinically obese boys and girls (standardized body mass index 3.07 ± 0.59, aged 8–18 years) were recruited. Anthropometric, body composition and cardiometabolic risk factors were measured pre- and post-weight loss.

Results

There were highly significant reductions in anthropometric, body composition and cardiometabolic risk factors. Triglyceride change was positively correlated with LDL peak particle density and percentage LDL pattern B changes (relative abundance of small, dense LDL particles). Multiple regression analyses showed that changes in triglyceride concentration accounted for between 24 and 18% of the variance in LDL peak particle density and percentage LDL pattern B change, respectively. Changes in waist circumference and insulin sensitivity did not predict these changes in LDL characteristics.

Conclusion

Acute and highly significant weight loss significantly decreased LDL peak particle density and percentage LDL pattern B. The change in triglycerides was a strong predictor of LDL peak particle density and percentage LDL pattern B change.

 

2.199           Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Brindley, M.A., Plattet, P. and Plemper, R.K.

PNAS, 111(36), E3795-E3804 (2014)

 

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry.

 

2.200           Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability

Perrault, S.D. and Shih, W.M.

ACSNano, 8(5), 5132-5140 (2014)

 

DNA nanotechnology enables engineering of molecular-scale devices with exquisite control over geometry and site-specific functionalization. This capability promises compelling advantages in advancing nanomedicine; nevertheless, instability in biological environments and innate immune activation remain as obstacles for in vivo application. Natural particle systems (i.e., viruses) have evolved mechanisms to maintain structural integrity and avoid immune recognition during infection, including encapsulation of their genome and protein capsid shell in a lipid envelope. Here we introduce virus-inspired enveloped DNA nanostructures as a design strategy for biomedical applications. Achieving a high yield of tightly wrapped unilamellar nanostructures, mimicking the morphology of enveloped virus particles, required precise control over the density of attached lipid conjugates and was achieved at 1 per 180 nm². Envelopment of DNA nanostructures in PEGylated lipid bilayers conferred protection against nuclease digestion. Immune activation was decreased 2 orders of magnitude below controls, and pharmacokinetic bioavailability improved by a factor of 17. By establishing a design strategy suitable for biomedical applications, we have provided a platform for the engineering of sophisticated, translation-ready DNA nanodevices.

 

2.201           Abstract 433: Examination of Factors Affecting the Association of PCSK9 With Low-Density Lipoprotein Particles in Human Plasma

Golder, M., Sarkar, S., Kosenko, T., McPherson, R. and Lagace, T.A.

Arterioscler. Thromb. Vasc. Biol., 34:A433 (2014)

 

Rationale: We have previously shown that a substantial proportion of plasma PCSK9 (30-40%) is associated with LDL particles in normolipidemic subjects. Cellular assays show that LDL-bound PCSK9 is less active for binding to cell surface LDLRs. Therefore, the ability of circulating PCSK9 to direct LDLR degradation in liver could be regulated by plasma LDL levels. In addition, LDL subspecies may have altered abilities in binding PCSK9. We have mapped the LDL binding region to a short stretch of amino acids (aa 31-52) in the PCSK9 prodomain. It is unknown whether a common loss-of-function PCSK9 mutation (R46L) within this region affects LDL binding.

Objective: To determine whether plasma PCSK9 distribution (LDL-bound versus unbound) is affected in hypercholesterolemic subjects. To further characterize the interaction of PCSK9 and LDL, we investigated the interaction of PCSK9 with two subspecies of LDL - large, buoyant LDL (LBLDL; d=1.019-1.044 g/ml) and small, dense LDL (SDLDL; d=1.044-1.063 g/ml). Additionally, we investigated the effect of the R46L PCSK9 mutation on the LDL binding affinity of PCSK9.

Methods and Results: We used flotation ultracentrifugation in Optiprep density gradients to fractionate human plasma samples followed by immunoprecipitation and western blot to quantify PCSK9 distribution in LDL and non-LDL fractions. In a pilot study, the proportion of total plasma PCSK9 in the LDL fraction was increased from 38±5% to 57±3% (N=6) in hypercholesterolemic subjects (LDL>4.9mM, TG<2.3mM) versus normal controls (LDL<3 mM, TG<2.3mM). Saturation binding assays showed that SDLDL bound PCSK9 with lower affinity (Kd = 361.9 nM) than LDLDL (Kd = 263.9 nM). Competition binding assays determined that recombinant purified PCSK9-R46L secreted from HEK293 cells did not bind to isolated LDL with significantly altered affinity compared to wild-type PCSK9.

Conclusion: Our preliminary results indicate that plasma PCSK9 distribution is altered in hypercholesterolemia, with an increased proportion of total PCSK9 bound to LDL particles. Our in vitro results suggest that circulating small, dense LDL may bind more poorly to PCSK9 than larger LDL subspecies.

 

2.202           Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Ganini, D. and Mason, R.P.

Free Radical Biology and Medicine, 76, 61-68 (2014)

 

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.

 

2.203           MPV17L2 is required for ribosome assembly in mitochondria

Rosa, I.D., Durigon, R., Pearce, S.F., Rorbach, J., Hirst, E.M.A., Vidoni, S., Reyes, A., Brea-Calvo, G., Minczuk, M., Woellhaf, M.W., Herrmann, J.M., Huynen, M.A., Holt, I.J. and Spinazzola, A.

Nucleic Acids Res., 42(13), 8500-8515 (2014)

 

MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ0 cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.

 

2.204           Transcriptomic characterization of short duration endoplasmic reticulum stress on cultured human proximal tubule cells

Zhang, Y., barati, M., Munoz, I., Li, M., Wilkey, D., Rouchka, E.and Merchant, M.

BMC Bioformatics, 15 (Suppl 10) P5 (2014)

 

Stress granules (SG) are formed as collections of protein and RNA (ribonucleoprotein structures) and continuously assembled/disassembled in response to stresses such as heat, osmotic, or oxidant stress; representing an attempt to survive the stress through salvage of important proteins and RNA. Recent research suggests diabetic nephropathy (DN) may change or alter SG biology and in conditions that model DN may involve the receptor for activated C-kinases (RACK1). The incorporation of RACK1 into stress granules may down-regulate programmed cell death and further may impart the ability to scaffold to and sequester key signaling proteins to affect cell survival or death. We hypothesized that the inappropriate or dysregulated scaffolding of proteins or RNA into stress granules may be of importance in the development of diabetic nephropathy. The goal of this study is to qualitatively and semi-quantitatively characterize the effects of cell culture conditions modeling diabetes and ER stress in conjunction with over-expression studies of SG stabilizing proteins on RNA transcripts.

 

2.205           A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Chu, N.K., Shabbir, W., Bove-Fwenderson, E., Araman, C., Lemmens-Gruber, R., harris, D.A. and Becker, C.F.W.

1. Biol. Chem., 289, 30144-30160 (2014)

 

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP^C into pathogenic PrP^Sc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

 

2.206           RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis

Magalska, A., Schellhaus, a.K., Moreno-Andres. D., Zanini, F., Schooley, A., Sachdev, R., Schwarz, H., Madlung, J. and Antonin, W.

Developmental Cell, 31(3), 305-318 (2014)

 

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

 

2.207           Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

Fukuhara, T., Wada, M., Nakamura, S., Ono, C., Shiokawa, M., yamamoto, S., Motomura, T., Okamoto, T., Okuzaki, D., Yamamoto, M., Saito, I., Wakita, T., Koike, K. and matsuura, Y.

PloS Pathogens, 10(12), e1004534 (2014)

 

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

 

2.208           Cytotoxicity of Human Endogenous Retrovirus K–Specific T Cells toward Autologous Ovarian Cancer Cells

Rycaj, K., Plummer, J.B., Yin, B., Li, M., Garza, J., Radvanyi, L., Ramondetta, L.M., Lin, K., Johanning, G.L., Tang, D.G. and Wang-Johanning, F.

Clin. Cancer Res., 21(2), 471-483 (2015)

 

Purpose: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells.

Experimental Design: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors.

Results: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K–specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFNγ production, and HERV-K–specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted.

Conclusion: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer.

 

2.209           Atomic Structure of T6SS Reveals Interlaced Array Essential to Function

Clemens, D.L., Ge, P., Horwitz, M.A. and Zhou, Z.H.

Cell, 160, 940-951 (2015)

 

Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.

 

2.210           MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

Schuhmacher, J.S., Rossmann, F., Dempwollf, F., Knauer, C., Altegoer, f., Steinchen, W., Dörrich, A.K., Klingl, A., Stephen, M., Linne, U., Thormann, K.M. and Bange, G.

PNAS, 7(10), 3092-3097 (2015)

 

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

 

2.211           Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Cortesio, C.L., Lewellyn, E.B.and Drubin, D.G.

Mol. Biol. Cell, 26, 1509-1522 (2015)

 

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.

 

2.212           Prevalence of plasma small dense LDL is increased in obesity in a Thai population

Kulanuwar, S., Tungtrongchitr, R., Billington, D. and Davies, I.G.

Lipis in Health and Disease, 14:30 (2015)

 

Background

Plasma low density lipoprotein (LDL) particles vary in size, density, electrical charge and chemical composition. An increased presence of small dense LDL (sdLDL), along with raised triglyceride concentrations and decreased high density lipoprotein (HDL) cholesterol concentrations is commonly known as the atherogenic triad and has been observed in some cases of obesity, principally in Europe and America. This study examines the prevalence of sdLDL in the plasma of an obese (BMI ≥ 25 kg/m²) Thai population.

Methods

Plasma from fasted obese (n = 48) and non-obese (n = 16) Thai participants was subjected to density gradient ultracentrifugation in iodixanol to separate lipoproteins. Gradients were unloaded top-to-bottom into 20 fractions which were assayed for cholesterol, triglyceride, apo B and apo A-1 to identify lipoprotein types and subtypes.

Results

LDL cholesterol was subfractionated into LDL I + II (fractions 3–6, ρ = 1.021-1.033 g/ml) which was considered to represent large buoyant LDL (lbLDL), LDL III (fractions 7–9, ρ = 1.036-1.039 g/ml) which was considered to represent sdLDL, and, LDL IV (fractions 10–12, ρ = 1.044-1.051 g/ml) which was considered to represent very sdLDL. Concentrations of LDL III and IV were increased by 15-20% in obese participants whilst that of LDL I + II was concomitantly decreased by 10%. This was accompanied by a 50% increase in plasma triglyceride concentrations and 15% decrease in HDL cholesterol concentrations. Only 3/16 (19%) non-obese participants had a pattern B LDL cholesterol profile (peak density of >1.033 g/ml), whilst 28/48 (58%) obese participants were pattern B. When expressed as a fraction of the LDL concentration, total sdLDL (i.e. LDL III + IV) showed highly significant correlations to plasma triglyceride concentrations and the triglyceride/HDL cholesterol ratio.

Conclusions

The prevalence of sdLDL is increased in obesity in a Thai population such that they demonstrate a similar atherogenic triad to that previously observed in European and American populations.

 

2.213           Cry Protein Crystals: A Novel Platform for Protein Delivery

Nair, M.S., Lee, M.M., Bonnegarde-Bernard, A., Wallace, J.A., Dean, D.H., Ostrowski, M.C., Burry, R.W., Boyaka, P.N. and Chan, K.

PloS One, 10(6), e0127669 (2015)

 

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

 

2.214           Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity

Ysselstein, Joshi, M., Mishra, V., Grigg, A.M., Asiago, J.M., McCabe, G.P., Stanciu, L.A., Post, C.B. and Rochet, J-C.

Neurobiology of Disease, 79, 150-163 (2015)

 

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

 

2.215           A novel and rapid method for obtaining high titre intact prion strains from mammalian brain

Wenborn, A., terry, C., Gros, N., Joiner, S., D’Castro, L., Panico, S., Sells, J., Cronier, S., Linehan, J.M., Brandner, S., Saibil, H.R., Collinge, J. and Wadsworth, J.D.F.

Scientific Reports, 5:10062 (2015)

 

Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported. Using high-throughput cell-based prion bioassay to re-examine prion purification from first principles we now report the isolation of prion strains to exceptional levels of purity from small quantities of infected brain and demonstrate faithful retention of biological and biochemical strain properties. The method’s effectiveness and simplicity should facilitate its wide application and expedite structural studies of prions.

 

2.216           The Effect of n-3 Fatty Acids on Small Dense Low-Density Lipoproteins in Patients With End-Stage Renal Disease: A Randomized Placebo-Controlled Intervention Study

Sørensen, G.V.B., Svensson, M., Strandhave, C., Schmidt, E.B., Jørgensen, K.A. and Christensen, J.H.

1. Renal Nutrition, 25(4), 376-380 (2015)

 

Objective

Patients with end-stage renal disease (ESRD) have a high risk of cardiovascular disease. Small dense low-density lipoprotein (sdLDL) particles are particularly atherogenic. Marine n-3 polyunsaturated fatty acids (PUFA) may have a beneficial effect on numbers of sdLDL particles, and the aim of this study was to investigate the effect of n-3 PUFA on plasma levels of sdLDL in patients with ESRD.

Methods

ESRD patients with cardiovascular disease (n = 161) on chronic hemodialysis were randomized to treatment with 1.7 g of n-3 PUFA (n = 81) or 2 g of placebo (olive oil; n = 80) for 3 months. The study was double-blinded. Densities of LDL and percentages of sdLDL (sdLDL%) of total LDL were measured before and after intervention. On the basis of sdLDL%, patients were classified as having lipid pattern A, I (intermediate), or B defined by a successive increase in sdLDL concentration and decrease in lipid particle size.

Results

n-3 PUFAs significantly reduced triglycerides. However, LDL cholesterol remained unchanged. In the n-3 group, the LDL density did not change significantly during follow-up. Similarly, the LDL density remained unchanged in the placebo group. In the n-3 group, the sdLDL% was 34% at baseline and unchanged at follow-up. At baseline 71% had LDL pattern A, 9% had pattern I, and 20% had pattern B, and none of these patterns were significantly changed by n-3 PUFA supplementation.

Conclusion

Dietary supplementation with 1.7 g of n-3 PUFA had no effect on LDL density or sdLDL levels in patients with ESRD.

 

2.217           Co-option of Membrane Wounding Enables Virus Penetration into Cells

Luisoni, S., Suomalainen, M., Boucke, K., Grzybek, M., Coskun, U. and Greber, U.F.

Cell Host & Microbe, 18, 75-85 (201)

 

During cell entry, non-enveloped viruses undergo partial uncoating to expose membrane lytic proteins for gaining access to the cytoplasm. We report that adenovirus uses membrane piercing to induce and hijack cellular wound removal processes that facilitate further membrane disruption and infection. Incoming adenovirus stimulates calcium influx and lysosomal exocytosis, a membrane repair mechanism resulting in release of acid sphingomyelinase (ASMase) and degradation of sphingomyelin to ceramide lipids in the plasma membrane. Lysosomal exocytosis is triggered by small plasma membrane lesions induced by the viral membrane lytic protein-VI, which is exposed upon mechanical cues from virus receptors, followed by virus endocytosis into leaky endosomes. Chemical inhibition or RNA interference of ASMase slows virus endocytosis, inhibits virus escape to the cytosol, and reduces infection. Ceramide enhances binding of protein-VI to lipid membranes and protein-VI-induced membrane rupture. Thus, adenovirus uses a positive feedback loop between virus uncoating and lipid signaling for efficient membrane penetration.

 

2.218           Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages

De Backer, L., Naessens, t., De Koker, S., Zagato, E., Demeester, J., Grooten, J., De Smedt, S.C. and Raemdonck, K.

1. Controlled Release, 217, 53-63 (2015)

 

The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~ 1 mg kg^− 1 siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

 

2.219           QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers

Brener, O. et al

Scientific Reports, 5:13222 (2015)

 

Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.

 

2.220           Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert, G.E., Novakovic, V.A., Shi, J., Rasmussen, J. and Pipe, S.W.

Blood, 126(10), 1237-1244 (2015)

 

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the α_IIbβ₃ integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

 

2.221           Structure-based drug design identifies polythiophenes as antiprion compounds

Herrmann, U.S. et al

Science Translational Medicine, 7(299), 299ra123 (2015)

 

In a mouse model of prion disease, Herrmann et al. evaluated the therapeutic efficacy of luminescent conjugated polythiophenes (LCPs), which are molecules with a high affinity for ordered protein aggregates. Intracerebral administration of LCPs into prion-infected mice using osmotic pumps increased survival. Solid-state nuclear magnetic resonance and in silico binding studies of LCPs to simplified model fibrils allowed the authors to define structural rules, which they then used for the design of LCPs with superior prophylactic and therapeutic potency. The new work demonstrates the feasibility of rational drug design for developing therapeutics to treat prion diseases.

 

2.222           Nutritional values and bioactive components of under-utilised vegetables consumed by indigenous people in Malaysia

Wahab, N.A., Ahdan, R., Aufa, Z.A., Kong, K.W., Johar, M.H., Shariff, Z.M. and ismail, A.

1. Sci. Food Agric., 95(13), 2704-2711 (2015)

 

BACKGROUND

Diverse plants species in the forest remain under-utilised and they are mainly consumed only by local people. However, increasing issues in food security prompted the present study, which explores the nutritional and antioxidant aspects of Malaysian under-utilised vegetables. The studied vegetables were Paku Nyai (Stenochlaena palustris), Cemperai (Champereia manillana), Maman Pasir (Cleome viscose), Dudung (Erechtites valerianifolia) and Semambuk (Ardisia pendula).

RESULTS

Overall, these vegetables exhibited a low proximal content but they were high in vitamin C [7.07–1263 mg kg⁻¹ edible fresh sample (EFS)] and β-carotene content (18.4–43.9 mg kg⁻¹ kg⁻¹ EFS). Cemperai had the highest calcium content (565 mg kg⁻¹ EFS), whereas Semambuk had the highest total phenolic content [28.21 g gallic acid equivalents kg⁻¹ edible dried sample (EDS)] and antioxidant activity (86.1%) measured using β-carotene bleaching assay. Maman Pasir contained the highest total flavonoid content (39.99 g CE kg⁻¹ EDS) and 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity (82.2%). The extracts of these vegetables had significantly prevented the oxidation of haemoglobin and low-density lipoprotein, which yielded a reduced production of malondialdehyde.

CONCLUSION

Semambuk and Maman Pasir are potent to be used as new food and functional food sources as they are rich in nutrients and antioxidants. © 2014 Society of Chemical Industry.

 

2.223           Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

Gerhold, J.M., Cansiz-Arda, S., Löhmus, M., Engberg, O., Reyes, A., van Rennes, H., Sanz, A., Holt, I.J., Cooper, H.M. and Spelbrink, J.N.

Scientific Reports, 5:15292 (2015)

 

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

 

2.224           α-Tocopherol bioavailability is lower in adults with metabolic syndrome regardless of dairy fat co-ingestion: a randomized, double-blind, crossover trial

Mah, E., Sapper, T.., Chitchumroonchokchai, C., Failla, M.L., Schill, K.E., Clinton, S.K., Bobe, G., Traber, M.G. and Bruno, R.S.

Am. J. Clin. Nutr., 102(5), 1070-1080 (2015)

 

Background: Increasing dietary fat intake is expected to improve α-tocopherol bioavailability, which could be beneficial for improving α-tocopherol status, especially in cohorts at high cardiometabolic risk who fail to meet dietary α-tocopherol requirements.

Objective: Our objective was to assess dose-dependent effects of dairy fat and metabolic syndrome (MetS) health status on α-tocopherol pharmacokinetics in plasma and lipoproteins.

Design: A randomized, crossover, double-blind study was conducted in healthy and MetS adults (n = 10/group) who ingested encapsulated hexadeuterium-labeled (d₆)–RRR–α-tocopherol (15 mg) with 240 mL nonfat (0.2 g fat), reduced-fat (4.8 g fat), or whole (7.9 g fat) milk before blood collection at regular intervals for 72 h.

Results: Compared with healthy participants, those with MetS had lower (P < 0.05) baseline plasma α-tocopherol (μmol/mmol lipid) and greater oxidized low-density lipoprotein (LDL), interleukin (IL)–6, IL-10, and C-reactive protein. Regardless of health status, d₆–α-tocopherol bioavailability was unaffected by increasing amounts of dairy fat provided by milk beverages, but MetS participants had lower estimated d₆–α-tocopherol absorption (±SEM) than did healthy participants (26.1% ± 1.0% compared with 29.5% ± 1.1%). They also had lower plasma d₆–α-tocopherol AUC from 0 to 72 h, as well as maximal concentrations (C_max: 2.04 ± 0.14 compared with 2.73 ± 0.18 μmol/L) and slower rates of plasma disappearance but similar times to C_max. MetS participants had lower d₆–α-tocopherol AUC from t = 0–12 h (AUC_{0–t final}) in lipoprotein fractions [chylomicron, very-low-density lipoprotein (VLDL), LDL, high-density lipoprotein]. Percentages of d₆–α-tocopherol AUC_{0–t final} in both the chylomicron (r = −0.46 to −0.52) and VLDL (r = −0.49 to −0.68) fractions were inversely correlated with oxidized LDL, IL-10, IL-6, and C-reactive protein.

Conclusions: At dietary intakes equivalent to the Recommended Dietary Allowance, α-tocopherol bioavailability is unaffected by dairy fat quantity but is lower in MetS adults, potentially because of greater inflammation and oxidative stress that limits small intestinal α-tocopherol absorption and/or impairs hepatic α-tocopherol trafficking. These findings support higher dietary α-tocopherol requirements for MetS adults. This trial was registered at www.clinicaltrials.gov as NCT01787591.

 

2.225           Surface acoustic wave controlled integrated band-pass filter

Skowronek, V., Rambach, R.W. and Franke, T.

Microfluid. Nanofluid., 19(2), 335-341 (2015)

 

We introduce a microfluidic band-pass filter for particles that is fully integrated in a polydimethylsiloxane-based microchannel device. This acoustic filter allows a continuous and label-free separation of particles. To demonstrate the functionality, mixtures of particles with different sizes are exposed to propagating surface acoustic waves generated by two laterally displaced interdigitated transducers, one on each side of the microchannel. Dependent on the frequency used, a specific size or even a size range of particles can be extracted. We sort particles of sizes of ~1–10 µm and estimate the size resolution to be smaller than ∆r < 0.88 µm. We examine the performance of the device and achieve a throughput of ~10⁵ particles/s with an efficiency as high as 99 %.

 

2.226           Hitchhiking nanoparticles: Reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes

Wayteck, L., Dewitte, H., De Backer, L., Breckpot, K., Demeester, J., De Smedt, S.C. and Raemdonck, K.

Biomaterials, 77, 143-154 (2016)

 

Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8⁺ T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8⁺ T cells.

 

2.227           In vitro reconstitution of B cell receptor–antigen interactions to evaluate potential vaccine candidates

Weaver, G.C., Villar, R.F., Kanekiyo, M., Nabel, G.J., Mascola, J.R. and Lingwood, D.

Nature Protocols, 11(2), 193-213 (2016)

 

Predicting immune responses before vaccination is challenging because of the complexity of the governing parameters. Nevertheless, recent work has shown that B cell receptor (BCR)-antigen engagement in vitro can prove a powerful means of informing the design of antibody-based vaccines. We have developed this principle into a two-phased immunogen evaluation pipeline to rank-order vaccine candidates. In phase 1, recombinant antigens are screened for reactivity to the germline precursors that produce the antibody responses of interest. To both mimic the architecture of initial antigen engagement and facilitate rapid immunogen screening, these antibodies are expressed as membrane-anchored IgM (mIgM) in 293F indicator cells. In phase 2, the binding hits are multimerized by nanoparticle or proteoliposome display, and they are evaluated for BCR triggering in an engineered B cell line displaying the IgM sequences of interest. Key developments that complement existing methodology in this area include the following: (i) introduction of a high-throughput screening step before evaluation of more time-intensive BCR-triggering analyses; (ii) generalizable multivalent antigen-display platforms needed for BCR activation; and (iii) engineered use of a human B cell line that does not display endogenous antibody, but only ectopically expressed BCR sequences of interest. Through this pipeline, the capacity to initiate favorable antibody responses is evaluated. The entire protocol can be completed within 2.5 months.

 

2.228           Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma

Kroiss, M., Plonne, D., Kendl, S., Schirmer, D., Ronchi, C.L., Schirbel, A., Zink, M., Lapa, C., Klinker, H., Fassnacht, M., Heinz, W and Sbiera, S.

Eur. J. Endocrinol., 174(3), 343-353 (2016)

 

Objective Oral mitotane (o,p′-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC).

Aim Serum mitotane concentrations >14 mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p′-dichlorodiphenylacetic acid (o,p′-DDA) and o,p′-dichlorodiphenyldichloroethane (o,p′-DDE).

Design Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI–H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins.

Results Chyle of the index patient contained 197 mg/ml mitotane, 53 mg/ml o,p′-DDA, and 51 mg/l o,p′-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction.

Discussion Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p′-DDE was similarly distributed, but 87.9±4.2% of o,p′-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI–H295 cells and reduced ER stress marker gene expression.

Conclusion Mitotane absorption involves chylomicron binding. High concentrations of o,p′-DDA and o,p′-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

 

 

2.229           Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Rudolph, S., Klein, A.N., Tusche, M., Schlosser, C., Elfgen, A., Brener, O, Teunissen, C., Gremer, L., Funke, S.A., Kutzsche, J. and Willbold, D.

PloS One, 11(2), e0147470 (2016)

 

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.

 

2.230           Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect

Hörl, G., Froehlich, H., Fersti, U., ledinski, G., Binder, J., Cvirn, G., Stojakovic, T., Trauner, M., Koidl, C., Tafeit, E., Amrein, K., Scharnagi, H., Jürgens, G. and Hallström, S.

PloS One, 11(2), e0148210 (2016)

 

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.

 

2.231           Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis

Lee, K-W. and Bogenhagen, D.F.

Methods in Mol. Biol., 1351, 67-79 (2016)

 

Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA–protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.

 

 

 

2.232           Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

Czarnota, A., Tyborowska, J., Peszynska-Sularz, G., Growatzka, B., Bienkowska-Szewczyk, K. and Grzyb, K.

Microb. Cell Fact., 15:62 (2016)

 

Background

Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2–3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of sHBsAg.

Results

The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412–425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes.

Conclusions

For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

 

2.233           Hepatitis C Virus-Induced Degradation of Cell Death-Inducing DFFA-Like Effector B Leads to Hepatic Lipid Dysregulation

Lee, E.M., Alsagheir, A., Wu, X., Hammack, C., Mclaughlan, J., Watanabe, N., Wakita, T., Kneteman, N.M., Douglas, D.N. and Tang, H.

1. Virol., 90(8), 4174-4185 (2016)

 

Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB.

 

2.234           Increased Presence of Remnant Lipoprotein Cholesterol in The Hdl of Diabetic Subjects

Gonzalez, M., Heras, M., Rosales, R., Guardiola, M., Plana, N., Vallve, J.C., Masana, L. and Ribalta, J.

Ann. Clin. Lab. Sci., 46(2), 229 (2016)

 

The atherogenic dyslipidemia associated with type II diabetes (T2DM) is characterized by elevated fasting triglycerides (TG) and remnant lipoproteins (RL) as well as small and dense low-density lipoprotein (sdLDL) particles and low high-density lipoprotein cholesterol (HDLc) levels [1]. Epidemiological studies have demonstrated that both fasting and non-fasting hyperlipidemia are important risk factors for atherosclerosis and cardiovascular events [2]. In the non-fasting state, the lipid profile is characterized by the accumulation of RL, a heterogeneous group of catabolized hepatic and intestinal lipoproteins, which differ in size, density, electrophoretic mobility, composition, and receptor affinity [3]. RL are considered an independent risk factor in cardiovascular disease [4–6]. The analytical determination of RL is not straightforward. Measurements of circulating apolipoprotein (apo) B48 estimate postprandial remnant lipoproteins [7]. On the other hand, the remnant-like particle cholesterol (RLPc) fraction obtained by immunchromatography identifies a wider spectrum of lipoproteins, ranging from VLDL to HDL, that cannot effectively bind to antibodies against apoA1 and apoB100 [8]. The RLPs density range varies according to the population studied; for instance, in normolipidemic subjects, RLPc is normally undetectable in the IDL fraction [5]. However, in diabetic or dysbetalipoproteinemic patients, the IDL can be found in the RLPc fraction [9].

The aim of this study is two-fold: 1) To analyze which lipoproteins have RLPs and how this varies between normolipidemic and T2DM patients; 2) To test how this distribution varies postprandially in normolipidemic subjects with iodixanol gradient ultracentrifugation, a method that allows for more accurate subfraction separation permitting the quantification of up to 21 lipoprotein subclasses [10].

 

2.235           Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

Klein, A.-N., Ziehm, T., Tusche, M., Buitenhuis, J., bartnik, D., Boeddrich, A., Wiglenda, T., Wanker, E., Funke, S.A., Brener, O., Gremer, L., Kutzsche, J. and Willbold, D.

PloS One, 11(4), e0153035 (2016)

 

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

 

2.236           A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion

Xu, W., Nathwani, B., Lin, C., Wang, J., karatekin, E., Pincet, F., Shih, W. and Rothman, J.E.

J. Am. Chem. Soc., 138(13), 4439-4447 (2016)

 

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.

 

2.237           Ex vivo mammalian prions are formed of paired double helical prion protein fibrils

Terry, C., Wenborn, A., Gros, N., Sells, J., Joiner, S., Hosszu, L.P., tattum, M.H., Panico, S., Clare, D.K., Collinge, J., Saibil, H.R. and Wadsworth, J.D.F.

Open Biology, 6, 160035 (2016)

 

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

 

2.238           Isolation of high density lipoproteins in ovine follicular and oviductal fluid

Bernecic, N.C., Gadella, B.M., de Graaf, S.P. and Leahy, T.

Animal Reproduction Science, 169, 121.122 (2016)

 

Capacitation is a key maturation process in spermatozoa that is vital for successful fertilisation. Following the initiation of this process, cholesterol is lost from the sperm plasma membrane via a series of regulated mechanisms which then permits appropriate sperm binding to the zona pellucida and the induction of the acrosome reaction. High density lipoproteins (HDL) have been shown to play a key role in cholesterol efflux in somatic cells but their physiological role in sperm cholesterol efflux is uncertain. The objective of this study was to isolate and identify HDL in ovine follicular and oviductal fluid for potential use in media that supports ram sperm capacitation in vitro.

Density gradients were generated by diluting Iodixanol (OptiPrep; Sigma Aldrich, Australia) to 20% (v/v) with follicular or oviductal fluid and then layering this mixture underneath equal volumes of 6% and 12.5% iodixanol in PBS (v/v). Samples were centrifuged (27,7320 × g at 16 °C for 5 h) and the resulting gradients were harvested into 10 fractions by aspiration. Proteins from each fraction were separated based on molecular weight using 1D SDS-PAGE. Liquid Chromatography Mass Spectrometry (LC–MS) was used to detect the presence of the major HDL apolipoprotein, apoA-I, in the relevant molecular weight band on the gels.

This proteomic approach confirmed the presence of apoA-I in fractions 7–10 of follicular fluid and fractions 8–10 of oviductal fluid. This indicates that HDLs in ovine follicular and oviductal fluid can be isolated using iodixanol and density ultracentrifugation. The establishment of this protocol in sheep provides an opportunity to investigate the function of HDLs as potential cholesterol acceptors from ram spermatozoa during in vitro capacitation, which will be the focus of future studies.

 

2.239           Self-assembly of size-controlled liposomes on DNA nanotemplates

Yang, Y., Wang, J., Shigematsu, H., Xu, W., Shih, W.M., Rothman, J.E. and Lin, C.

Nature Chem., 8(5), 476-483 (2016)

 

Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision.

 

2.240           PCSK9 Association With Lipoprotein(a)

Tavori, H., Christian, D., Minnier, J., Plubell, D., Shapiro, M.D., Yeang, C., Giunzioni, I., Croyal, M., Duell, P.B., Lambert, G., Tsimikas, S. and Fazio, S.

Circ. Res., 119, 29-35 (2016)

 

Rationale: Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein–like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B. Proprotein convertase subtilisin/kexin type 9 (PCSK9) selectively binds low-density lipoprotein; we hypothesized that it can also be associated with Lp(a) in plasma.

Objective: Characterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39–320 mg/dL) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via coexpression of human apo(a) and human apolipoprotein B).

Methods and Results: We show that PCSK9 is physically associated with Lp(a) in vivo using 3 different approaches: (1) analysis of Lp(a) fractions isolated by ultracentrifugation; (2) immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (3) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only, and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) versus low-density lipoprotein (1.7-fold increase) was seen in subjects with high Lp(a) and normal low-density lipoprotein. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels.

Conclusions: Our results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

 

2.241           TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia

Yeh, F.L., Wang, Y., Tom, I., Gonzales, L.C. and Sheng, M.

Neuron, 91, 328-340 (2016)

 

Genetic variants of TREM2, a protein expressed selectively by microglia in the brain, are associated with Alzheimer’s disease (AD). Starting from an unbiased protein microarray screen, we identified a set of lipoprotein particles (including LDL) and apolipoproteins (including CLU/APOJ and APOE) as ligands of TREM2. Binding of these ligands by TREM2 was abolished or reduced by disease-associated mutations. Overexpression of wild-type TREM2 was sufficient to enhance uptake of LDL, CLU, and APOE in heterologous cells, whereas TREM2 disease variants were impaired in this activity. Trem2 knockout microglia showed reduced internalization of LDL and CLU. β-amyloid (Aβ) binds to lipoproteins and this complex is efficiently taken up by microglia in a TREM2-dependent fashion. Uptake of Aβ-lipoprotein complexes was reduced in macrophages from human subjects carrying a TREM2 AD variant. These data link three genetic risk factors for AD and reveal a possible mechanism by which mutant TREM2 increases risk of AD.

 

2.242           Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Fogeron, M-L. et al

1. Biomol. NMR, 65(2), 87-98 (2016)

 

We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [²H,¹³C,¹⁵N]-labeled protein are shown to yield narrow ¹³C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

 

2.243           Eicosapentaenoic Acid Inhibits Oxidation of ApoB-containing Lipoprotein Particles of Different Size In Vitro When Administered Alone or in Combination With Atorvastatin Active Metabolit Compared With Other Triglyceride-lowering Agents

Mason, R.P., Sherratt, S.C.R. and Jacob, R.F.

1. Cardiovasc. Pharmacol., 68(1), 33-40 (2016)

 

Eicosapentaenoic acid (EPA) is a triglyceride-lowering agent that reduces circulating levels of the apolipoprotein B (apoB)-containing lipoprotein particles small dense low-density lipoprotein (sdLDL), very–low-density lipoprotein (VLDL), and oxidized low-density lipoprotein (LDL). These benefits may result from the direct antioxidant effects of EPA. To investigate this potential mechanism, these particles were isolated from human plasma, preincubated with EPA in the absence or presence of atorvastatin (active) metabolite, and subjected to copper-initiated oxidation. Lipid oxidation was measured as a function of thiobarbituric acid reactive substances formation. EPA inhibited sdLDL (IC50 ∼2.0 μM) and LDL oxidation (IC50 ∼2.5 μM) in a dose-dependent manner. Greater antioxidant potency was observed for EPA in VLDL. EPA inhibition was enhanced when combined with atorvastatin metabolite at low equimolar concentrations. Other triglyceride-lowering agents (fenofibrate, niacin, and gemfibrozil) and vitamin E did not significantly affect sdLDL, LDL, or VLDL oxidation compared with vehicle-treated controls. Docosahexaenoic acid was also found to inhibit oxidation in these particles but over a shorter time period than EPA. These data support recent clinical findings and suggest that EPA has direct antioxidant benefits in various apoB-containing subfractions that are more pronounced than those of other triglyceride-lowering agents and docosahexaenoic acid.

 

2.244           Increase of Positive Net Charge and Conformational Rigidity Enhances the Efficacy of d-Enantiomeric Peptides Designed to Eliminate Cytotoxic Aβ Species

Ziehm, T., Brener, O., van Groen, T., Kadish, I., Frenzel, D., Tusche, M., Kutzsche, J., Reiss, K., Gremer, L., Nagel-Steger, L. and Willbold, D.

ACS Chem. Neurosci., 7(8), 1088-1096 (2016)

 

Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aβ) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3_COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3_COOH were designed: C-terminally amidated linear D3 (D3_CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3_CONH2. These four compounds were compared to each other according to their binding affinities to Aβ(1–42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aβ(1–42) oligomer induced neurotoxicity. D3_CONH2 and cD3r versions with equally increased net charge showed superior properties over D3_COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aβ targeting compound D3.

 

2.245           14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

Li, T. and Paudel, H.K.

PloS One, 11(8), e0160635 (2016)

 

Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau

 

2.246           Hepatitis C virus (HCV) and atherosclerosis risk: A role for low-density immune complexes?

Bassendine, M., Nielsen, S. and Neely, D.

Atherosclerosis, 252, e206 (2016)

 

Objectives: HCV is associated with an increased risk of atherosclerosis, despite inducing a favourable lipid profile with accompanying low classical risk score. HCV lipo-viral particles and sub-viral particles are found in the low–density fraction (LDF) of blood (<1.08 g/ml) associated with apoB-containing lipoproteins. The aim of this study was to examine the proteome of LDF in chronic HCV, compared to non-HCV control.

Methods: Proteins in iodixanol fractions of blood were separated on SDS PAGE; 14 bands excised from the gel were analysed by MALDI TOF. Peptides generated by trypsin digestion were analysed by mass spectrometry. Protein bands were identified using the peptide mass fingerprint data and the Mascot search engine program, searched against NCBI protein.

Results: Several protein bands were detected in the LDF only in HCV patients. After identification of protein bands by mass spectrometry, lanes were scanned to produce an intensity profile. Peaks corresponding to proteins identified by mass spectrometry were labelled indicating that four proteins, IgM heavy chain, IgG3 heavy chain, IgG1 heavy chain and Kappa light chain were present only in LDF purified from HCV patients.

 

2.247           Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins

Thomas, S.E. and Harrison, E.H.

1. Lipid Res., 57, 1865-1878 (2016)

 

The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin.

 

2.248           Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation

Streich, C., Akkari, L., Decker, C., Bormann., J., Rehbock, C., Müller-Sciffmann, A., Carlsson Niemayer, F., Nagel-Steger, L., Wilbold, d., Sacca, B., Korth, C., Schrader, T. and Barcikowski, S.

ACS Nano, 10(8), 7582-7597 (2016)

 

Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody–antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aβ-specific small molecules to inhibit Aβ peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aβ are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for β-sheet content, and inhibition of cellular Aβ excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer’s disease.

 

2.249           Size Determination of a Liposomal Drug by Small-Angle X-ray Scattering Using Continuous Contrast Variation

Garcia-Diez, R., Gollwitzer, C., Krumrey, M. and Varga, Z.

Langmuir, 32(3), 772-778 (2016)

 

The continuously growing complexity of nanodrugs urges for complementary characterization techniques which can elude the current limitations. In this paper, the applicability of continuous contrast variation in small-angle X-ray scattering (SAXS) for the accurate size determination of a complex nanocarrier is demonstrated on the example of PEGylated liposomal doxorubicin (Caelyx). The mean size and average electron density of Caelyx was determined by SAXS using a gradient of aqueous iodixanol (Optiprep), an iso-osmolar suspending medium. The study is focused on the isoscattering point position and the analysis of the Guinier region of the scattering curves recorded at different solvent densities. An average diameter of (69 ± 5) nm and electron density of (346.2 ± 1.2) nm^–3 were determined for the liposomal formulation of doxorubicin. The response of the liposomal nanocarrier to increasing solvent osmolality and the structure of the liposome-encapsulated doxorubicin after the osmotic shrinkage of the liposome are evaluated with sucrose contrast variation in SAXS and wide-angle X-ray scattering (WAXS). In the case of using sucrose as contrast agent, a clear osmolality threshold at 670 mOsm kg^–1 was observed, above which the liposomal drug carriers start to shrink, though preserving the intraliposomal doxorubicin structure. The average size obtained by this technique is smaller than the value measured by dynamic light scattering (DLS), though this difference is expected due to the hydrodynamic size of the PEG moieties attached to the liposomal surface, which are not probed with solvent contrast variation in SAXS. The advantages and drawbacks of the proposed technique are discussed in comparison to DLS, the most frequently used sizing method in nanomedicine.

 

2.250           High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-β to Ganglioside GM1 Containing Nanodiscs

Thomaier, M., Gremer, L., Dammers, C., Fabig, J., Neudecker, P. and Willbold, D.

Biochemistry, 55(48), 6662-6672 (2016)

 

The interaction of the amyloid-β protein (Aβ) with neuronal cell membranes plays a crucial role in Alzheimer’s disease. Aβ undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aβ interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aβ membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aβ. In particular, we set out to investigate whether the binding activity of GM1 to Aβ is specific for the assembly state of Aβ and compared the binding affinities of monomeric with oligomeric Aβ. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aβ(1–40) to GM1 containing nanodiscs, with dissociation constants, K_D, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aβ(1–40) (K_D of 24 nM to 49 nM) as well as of Aβ(1–42) (K_D of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aβ, neither oligomeric Aβ(1–40) nor oligomeric Aβ(1–42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aβ upon aggregation.

 

2.251           Plasma transport of ergocalciferol and cholecalciferol and their 25-hydroxylated metabolites in dairy cows

Hymøller, L. and Jensen, S.K.

Domestic Animal Endocrinology, 59, 44-52 (2017)

 

In cattle, there are 2 significant forms of vitamin D: ergocalciferol (ERG) from fungi on roughage and cholecalciferol (CHO) from vitamin supplements or endogenous synthesis in the skin. The hypothesis of the present study is that vitamin D from the 3 sources is transported in different plasma fractions in the body. This is hypothesized to explain the lower efficiency of ERG compared to CHO in securing a sufficient plasma status of 25-hydroxyvitamin D and explain the inefficient excretion of dietary CHO into milk compared to endogenous CHO. Twenty vitamin D–depleted cows were assigned to 5 treatments: D2, housed indoor and fed 625-μg/d (25.000 IU) ERG; D3, housed indoor and fed 625-μg/d CHO; D2+D3, housed indoor and fed 625-μg/d ERG and 625-μg/d CHO; SUN, let out for daily pasture to facilitate CHO synthesis from sunlight; and D2+SUN, fed 625-μg/d ERG and let out for daily pasture. Blood samples were taken twice weekly and plasma fractionated by ultracentrifugation into 3 fractions: light lipoprotein (LLP), heavy lipoprotein (HLP), and protein and analyzed for content of ERG and CHO and their liver derived metabolites 25-hydroxyergocalciferol (25ERG) and 25-hydroxycholecalciferol (25CHO), respectively. Liver biopsies were taken on the last day of the study to asses gene expression related to vitamin D metabolism. During 4 wk of study, the vitamin D status in plasma increased to 19.3 to 22.8 ng/mL 25ERG in ERG-treated cows with the highest concentration in D2 (P ≤ 0.05) and to 25.0 to 33.4 ng/mL 25CHO in pasture or CHO-treated cows with the highest concentration in SUN (P ≤ 0.01). In plasma fractions, CHO was mainly found in the HLP fraction, whereas 25CHO was almost exclusively found in the protein fraction, probably due to its reported high binding affinity to vitamin D–binding protein. About 70% to 90% of 25ERG was found in the protein fraction and the remaining 25ERG was found in HLP, whereas ERG was found in both HLP and LLP fractions. In liver tissue, the expression of vitamin D-25-hydroxylase was lower in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05) than that in the remaining groups, and the vitamin D receptor was expressed in the liver to a larger extent in D2+SUN than that in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05). In conclusion, different plasma transport mechanisms may explain the lower physiological efficiency of ERG compared to CHO in securing the vitamin D status in plasma but do not explain the lower efficiency of synthetic CHO compared to endogenous CHO from sunlight or UV light in securing a high CHO content in milk.

 

2.252           Omega-3 fatty acid fish oil dietary supplements contain saturated fats and oxidized lipids that may interfere with their intended biological benefits

Preston Mason, R. and Sherratt, A.C.R.

Biochem. Biophys. Res. Comm., 483, 425-429 (2017)

 

Widely available fish oil dietary supplements (DS) may contain fats and oxidized lipids in addition to the beneficial omega-3 fatty acids (OM3FAs) for which they are purchased. Little is known about the potential biological effects of these oxidized lipids. The objective of this study was to assess the fatty acid content, oxidation products, and biological effects of leading fish oil DS available in the United States. Three top-selling fish oil DS in the US were included in this analysis. Fatty acid composition was measured using gas chromatography. Lipid oxidation (primary and secondary products) was measured by spectroscopy in both DS and a prescription OM3FA product. OM3FAs were also isolated and concentrated from DS and were tested for the ability to inhibit copper-induced oxidation of human small dense low-density lipoprotein particles (sdLDL) in vitro. Fish oil DS were found to contain more than 30 different fatty acids, including 10 to 14 different saturated species comprising up to 36% of the total fatty acid content. Levels of OM3FAs also varied widely among DS (33%–79%). Primary (peroxide), secondary (anisidine), and total oxidation products exceeded maximum levels established by international standards of quality in the DS but not the prescription OM3FA product. Oxidation of sdLDL was inhibited by >95% (P < 0.001) with non-oxidized forms of OM3FA but not with OM3FAs isolated from DS, which were a mixture of oxidized and non-oxidized OM3FAs. These data indicate that levels of saturated fat and oxidized OM3FAs found in common DS may interfere with their intended/potential biological benefits.

 

2.253           Controlling the gastrointestinal fate of nutraceutical and pharmaceutical-enriched lipid nanoparticles: From mixed micelles to chylomicrons

Yao, M., McClements, D.J., Zhao, F., Craig, R.W. and Xiao, H.

NanoImpact, 5(1), 13-21 (2017)

 

The oral bioavailability of lipophilic bioactive compounds such as many pharmaceuticals and nutraceuticals can be enhanced using triacylglycerol-based lipid nanoparticle delivery systems. These digestible lipid nanoparticles are dissembled in the gastrointestinal tract to form mixed micelles that solubilize and transport the lipophilic bioactives to the intestinal epithelium cells where they are absorbed. In these cells, the lipid digestion products and bioactive agents contained within the mixed micelles are then packaged into biological lipid protein nanoparticles (e.g., chylomicrons) that are secreted into the lymph. In this study, we examined the influence of fatty acid type (i.e., oleic acid, linoleic acid, and linolenic acid) on the properties of mixed micelles, cellular lipid droplets, and lipoprotein nanoparticles, and on the bioavailability of a highly lipophilic nutraceutical: 5-demethylnobiletin (5DN). There were distinct differences in the structural properties of lipoprotein nanoparticles formed depending on fatty acid unsaturation. Oleic acid (C_18:1) was most effective in enhancing intestinal uptake of 5DN and led to the formation of the largest chylomicrons. Linoleic acid (C_18:2) and linolenic acid (C_18:3) also promoted intestinal uptake of 5DN and formation of chylomicrons, but they were less efficient than oleic acid. The metabolism of 5DN within the intestinal epithelium cells was greatly reduced when 5DN was incorporated into chylomicrons, presumably because they were isolated from metabolic enzymes in the cytoplasm. These results have important implications for the rational design of lipid nanoparticle-based delivery systems for lipophilic nutraceuticals and pharmaceuticals by targeting them to the lymphatic circulation.

 

2.254           Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

Chandrasekar, S. and Shan, S-o.

1. Biol. Chem., 292(1), 397-406 (2017)

 

The universally conserved signal recognition particle (SRP) co-translationally delivers newly synthesized membrane and secretory proteins to the target cellular membrane. The only exception is found in the chloroplast of green plants, where the chloroplast SRP (cpSRP) post-translationally targets light-harvesting chlorophyll a/b-binding proteins (LHCP) to the thylakoid membrane. The mechanism and regulation of this post-translational mode of targeting by cpSRP remain unclear. Using biochemical and biophysical methods, here we show that anionic phospholipids activate the cpSRP receptor cpFtsY to promote rapid and stable cpSRP54·cpFtsY complex assembly. Furthermore, the stromal domain of the Alb3 translocase binds with high affinity to and regulates GTP hydrolysis in the cpSRP54·cpFtsY complex, suggesting that cpFtsY is primarily responsible for initial recruitment of the targeting complex to Alb3. These results suggest a new model for the sequential recruitment, remodeling, and unloading of the targeting complex at membrane translocase sites in the post-translational cpSRP pathway.

 

2.255           Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

Haddad, J.G., Rouille, Y., Hanoulle, X., Descamps, V., Hamze, M., Dabbousi, F., Baumert, T.F., Duverlie, G., Lavie, M. and Dubuisson, J.

1. Virol., 91(8), e-00048-17 (2017)

 

Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.

 

2.256           Lipid Membrane Encapsulation of a 3D DNA Nano Octahedron

Perrault, S.D. and Shih, W.M.

Methods in Mol. Biol., 1500, 165-184 (2017)

 

Structural DNA nanotechnology methods such as DNA origami allow for the synthesis of highly precise nanometer-scale materials (Rothemund, Nature 440:297–302, 2006; Douglas et al., Nature 459:414–418, 2009). These offer compelling advantages for biomedical applications. Such materials can suffer from structural instability in biological environments due to denaturation and nuclease digestion (Hahn et al., ACS Nano 2014; Perrault and Shih, ACS Nano 8:5132–5140, 2014). Encapsulation of DNA nanostructures in a lipid membrane compartmentalizes them from their environment and prevents denaturation and nuclease digestion (Perrault and Shih, ACS Nano 8:5132–5140, 2014). Here, we describe the encapsulation of a 50 nm DNA nanostructure having the geometry of a wireframe octahedron in a phospholipid membrane containing poly-(ethylene glycol), resulting in biocompatible DNA nanostructures.

 

2.257           The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly

Donlin-Asp, P.G., Fallini, C., Campos, J., Chou, C.C., Merritt, C.C., Bassell, G.J. and Rossoll, W.

Cell Reports, 18, 1660-1673 (2017)

 

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3′ UTR zipcode region of β-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.

 

2.258           Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein

Leske, H., Hornemann, U., Zhu, C. et al

PloS One, 12(2), e0170503 (2017)

 

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrP^Sc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrP^Sc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrP^Sc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2–α2 loop of the cellular prion protein (PrP^C). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrP^C but expressing MoPrP^169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP^169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP^169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP^169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2–α2 loop of PrP^C affects the sensitivity of PrP^Sc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrP^C can be linked to a physicochemical property of the corresponding PrP^Sc.

 

2.259           CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

Hurwitz, S.N., Nkosi, D., Conlon, M.M., York, S.B., Liu, X., Tremblay, D.C. and Meckes, D.G.

1. Virol., 91(5), e02251-16 (2017)

 

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.

 

2.260           Post-translational modifications in PrP expand the conformational diversity of prions in vivo

Aguilar-Calvo, P., Xiao, X., Bett, C., Erana, H., Soldau, K., Castilla, J., Nilsson, K.P.R., Surewicz, W.K. and Sigurdson, C.J.

Scientific Reports, 7:43295 (2017)

 

Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPI-anchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of β-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in post-translational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

 

2.261           Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum

Ishikawa-Ankerhold, H.C., Daszkiewicz, W., Schleicher, M. and Müller-Taubenberger, A.

Scientific Reports, 7:40310 (2017)

 

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

 

2.262           Enhanced neuroinvasion by smaller, soluble prions

Bett, C., Lawrence, J., Kurt, T.D., Orru, C., Aguilar-Calvo, P., Kincaid, A.E., Surewicz, W.K., Caughey, B., Wu, C. and Sigurdson, C.J.

Acta Neuropathol.Commun., 5:32 (2017)

 

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

 

 

 

2.263           Multiple pathogen biomarker detection using an encoded bead array in droplet PCR

Rajeswari, P.K.P., Soderberg, L.M., Yacoub, A., Leijon, M., Andersson Svahn, H. and Joensson, H.N.

1. Microbiol. Methods, 139, 22-28 (2017)

 

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

 

2.264           Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Rouvinski, A. et al

Nature Communications, 8:15411 (2017)

 

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic ‘breathing’ of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.

 

2.265           Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin

Waldhart, A.N., Dykstra, H., Peck, A.S., Cantley, L.C., Graw, T.E. and Wu, N.

Cell Reports, 19, 2005-2013 (2017)

 

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.

 

2.266           Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

Oliveira, C. et al

Hepatology, 65(6), 1851-1864 (2017)

 

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection.

 

2.267           Heterogeneous Defect Domains in Single-Crystalline Hexagonal WS2

Jeong, H.Y., Jin, Y., Yun, S.J., Zhao, J., Baik, J., Keum, D.H., Lee, H.S. and Lee, Y.H.

Advanced Materials, 29:160543 (2017)

 

Single-crystalline monolayer hexagonal WS₂ is segmented into alternating triangular domains: sulfur-vacancy (SV)-rich and tungsten-vacancy (WV)-rich domains. The WV-rich domain with deep-trap states reveals an electron-dedoping effect, and the electron mobility and photoluminescence are lower than those of the SV-rich domain with shallow-donor states by one order of magnitude. The vacancy-induced strain and doping effects are investigated via Raman and scanning photoelectron microscopy.

 

2.268           Placing and shaping liposomes with reconfigurable DNA nanocages

Zhang, Z., Yang, Y., Pincet, F., Llaguno, M.C. and Lin, C.

Nature Chem., 9(7), 653-659 (2017)

 

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

 

2.269           Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

Diedrich, B., Rigbolt, K.T.G., Röring, M., herr, R., Kaeser-Pebernard, S., Gretzmeier, C., Murphy, R.F., Brummer, T. and Dengjel, J.

EMBO J., 36(5), 646-663 (2017)

 

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAF^V600E and wild‐type BRAF (BRAF^WT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAF^V600E resides in large complexes of higher molecular mass and activity, while BRAF^WT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐Ras^G12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAF^WT into large, active complexes, whereas pharmacological inhibition of BRAF^V600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

 

2.270           Impact of Lipid Phase on the Bioavailability of Vitamin E in Emulsion-Based Delivery Systems: Relative Importance of Bioaccessibility, Absorption, and Transformation

Yang, Y., Xiao, H. and McClements, D.J.

Agric. Food Chem., 65, 3946-3955 (2017)

 

A simulated gastrointestinal tract/Caco-2 cell culture model was used to investigate the effects of lipid phase type on vitamin E (VE) bioavailability. Oil-in-water emulsions fortified with α-tocopherol acetate were fabricated using a natural emulsifier (quillaja saponin) and long or medium chain triglycerides (LCTs or MCTs) as lipids. The impact of lipid type on VE bioaccessibility, absorption, and transformation was determined. VE bioaccessibility was greater for LCT (46%) than MCT (19%) due to greater solubilization in mixed micelles assembled from longer fatty acids. VE absorption by Caco-2 cells was similar for LCT (28%) and MCT (30%). The transformation of α-tocopherol acetate to α-tocopherol was higher for LCT (90%) than MCT (75%) due to differences in esterase accessibility to VE. Emulsion-based delivery systems formulated using LCT are therefore more suitable for encapsulating and delivering vitamin E than those formulated using MCT.

 

2.271           Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification

Wade, J.H., Jones, J.D., lenov, I., Riordan, C.M., Sligar, S.G. and Bailey, R.C.

Lab on a Chip, 17, 2951-2959 (2017)

 

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

 

2.272           Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick

Igel-Egalon, A., Moudjou, M., Martin, D., Busley, A., Knäpple, T., Herzog, L., Reine, F., Lepajova, N., Richard, C-A., Beringue, V. and Rezaei, H.

PloS Pathogens, 13(9), e1006557 (2017)

 

Mammalian prions, the pathogens that cause transmissible spongiform encephalopathies, propagate by self-perpetuating the structural information stored in the abnormally folded, aggregated conformer (PrP^Sc) of the host-encoded prion protein (PrP^C). To date, no structural model related to prion assembly organization satisfactorily describes how strain-specified structural information is encoded and by which mechanism this information is transferred to PrP^C. To achieve progress on this issue, we correlated the PrP^Sc quaternary structural transition from three distinct prion strains during unfolding and refolding with their templating activity. We reveal the existence of a mesoscopic organization in PrP^Sc through the packing of a highly stable oligomeric elementary subunit (suPrP), in which the strain structural determinant (SSD) is encoded. Once kinetically trapped, this elementary subunit reversibly loses all replicative information. We demonstrate that acquisition of the templating interface and infectivity requires structural rearrangement of suPrP, in concert with its condensation. The existence of such an elementary brick scales down the SSD support to a small oligomer and provide a basis of reflexion for prion templating process and propagation.

 

 

2.273           A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels

Khetarpal, S.A. et al

Nature Med., 23(9), 1086-1094 (2017)

 

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels1. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance2. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD3, 4, 5. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

 

2.274           Metalloprotease-mediated cleavage of PlexinD1 and its sequestration to actin rods in the motoneuron disease spinal muscular atrophy (SMA)

Rademacher, S., Verheijen, B.M., Hensel, N., Peters, M., Bora, G., Brandes, G., de Sa, R.V., Heidrich, N., Fischer, S., Brinkmann, H.,van  der Pol, W.L., Wirth, B., Pasterkamp, R.J. and Claus, P.

Hum. Mol. Genet., 26(20), 3946-3959 (2017)

 

Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly motoneurons, but receptor-ligand interactions leading to the dysregulation causing SMA are poorly understood. In this study, we analysed the role of the guidance receptor PlexinD1 in SMA pathogenesis. We showed that PlexinD1 is cleaved by metalloproteases in SMA and that this cleavage switches its function from an attractant to repellent. Moreover, we found that the PlexinD1 cleavage product binds to actin rods, pathological aggregate-like structures which had so far been described for age-related neurodegenerative diseases. Our data suggest a novel disease mechanism for SMA involving formation of actin rods as a molecular sink for a cleaved PlexinD1 fragment leading to dysregulation of receptor signaling.

 

Conclusions Our findings highlight a new mechanism in lipid metabolism regulation and interaction of the lipid metabolism with the HCV life cycle, which may be important for viral pathogenesis and might also be explored for antiviral therapy.

 

2.275           Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

Chen, D., Xiang, Q. and Nicholas, J.

1. Virol., 91(22), e00965-17 (2017)

 

Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase and signal transducer and activator of transcription signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of proapoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, preproteolytically processed procathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. We show here that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), which catalyzes monoglucosylation of N-glycans, and oppositely acting glucosidase II (GlucII), and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1, and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment.

 

2.276           Optimization of d-Peptides for Aβ Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aβ Oligomers

Klein, A.N., Ziehm, T., van Groen, T., Kadish, I., Elfgen, A., Tusche, M., Thomaier, M., Reiss, K., Brener, O., Gremer, l., Kutzsche, J. and Wilbold, D.

ACS Chem. Neurosci., 8, 1889-1900 (2017)

 

Amyloid-beta (Aβ) oligomers are thought to be causative for the development and progression of Alzheimer’s disease (AD). Starting from the Aβ oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aβ(1–42) to further enhance the Aβ oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aβ(1–42), eliminated Aβ(1–42) oligomers, inhibited Aβ(1–42) fibril formation, and reduced Aβ(1–42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aβ(1–42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aβ monomers and thereby induced elimination of Aβ oligomers is a suitable therapeutic strategy.

 

2.277           How We Make DNA Origami

Wagenbauer, K.F., Engelhardt, F.A.S., Stahl, E., Hechtl, V.K., Stömmer, P., Seebacher, F., Meregalli, L., Ketterer, P., Gerling, T. and Dietz, H.

ChemBioChem., 18(19), 1873-1885 (2017)

 

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.

 

2.278           The Aβ oligomer eliminating D-enantiomeric peptide RD2 improves cognition without changing plaque pathology

Van Groen, T., Schemmert, S., Brener, O., gremer, l., Ziehm, T., Tusche, m., nagel-Steger, L., Kadish, I., Schartmann, E., Elfgen, A., Jürgens, D., Willuweit, A., Kutzsche, J. and Willbold, D.

Scientific Reports, 7.16275 (2017)

 

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer’s disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the d-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aβ42. Our findings demonstrate that RD2 was significantly more efficient in Aβ oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aβ oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aβ oligomer elimination by RD2 treatment may be also beneficial for AD patients.

 

2.279           UNC-45a promotes myosin folding and stress fiber assembly

Lehtimäki, J.I., Fenix, A.M., Kotila, T.M., Balistreri, G., Paavolainen, L., varjosalo, M., Burnette, D.T. and Lappalainen, P.

J. Cell Biol., 216(12), 4053-4072 (2017)

 

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

 

2.280           A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Munoz-Montesino, C., Sizun, C., Moudjou, M., Herzog, L., Reine, F., Igel-Egalon, A., Barbereau, C., Chapuis, J., Ciric, D., Laude, H., Beringue, V., Rezaei, H. and Dron, M.

Prion, 11(1), 25-30 (2017)

 

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP^C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP^C nor for the formation of a stable prion structure.

 

2.281           Circulating ApoJ is closely associated with insulin resistance in human subjects

Seo, J.A., Kang, M-C., Ciaraldi, T.P., Kim, S.S., Park, K.S., Choe, C., hwang, W.H., Lim, D.M., Farr, O., Mantzoros, C., Henry, R. and Kim, Y-B.

Metabolism, 78, 155-166 (2018)

 

Objective

Insulin resistance is a major risk factor for type 2 diabetes. ApolipoproteinJ (ApoJ) has been implicated in altered pathophysiologic states including cardiovascular and Alzheimer's disease. However, the function of ApoJ in regulation of glucose homeostasis remains unclear. This study sought to determine whether serum ApoJ levels are associated with insulin resistance in human subjects and if they change after interventions that improve insulin sensitivity.

Methods

Serum ApoJ levels and insulin resistance status were assessed in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosiglitazone or metformin therapy on serum ApoJ levels and glucose disposal rate (GDR) during a hyperinsulinemic/euglycemic clamp were evaluated in a separate cohort of T2D subjects. Total ApoJ protein or that associated with the HDL and LDL fractions was measured by immunoblotting or ELISA.

Results

Fasting serum ApoJ levels were greatly elevated in T2D subjects (ND vs T2D; 100 ± 8.3 vs. 150.6 ± 8.5 AU, P < 0.0001). Circulating ApoJ levels strongly correlated with fasting glucose, fasting insulin, HOMA-IR, and BMI. ApoJ levels were significantly and independently associated with HOMA-IR, even after adjustment for age, sex, and BMI. Rosiglitazone treatment in T2D subjects resulted in a reduction in serum ApoJ levels (before vs. after treatment; 100 ± 13.9 vs. 77 ± 15.2 AU, P = 0.015), whereas metformin had no effect on ApoJ levels. The change in ApoJ levels during treatment was inversely associated with the change in GDR. Interestingly, ApoJ content in the LDL fraction was inversely associated with HOMA-IR.

Conclusion

Serum ApoJ levels are closely correlated with the magnitude of insulin resistance regardless of obesity, and decrease along with improvement of insulin resistance in response only to rosiglitazone in type 2 diabetes.

 

2.282           Eicosapentaenoic acid inhibits oxidation of high density lipoprotein particles in a manner distinct from docosahexaenoic acid

Sherratt, S.C.R. and mason, R.P.

Biochem. Biophys. Res. Comm., 496, 315-338 (2018)

 

The omega-3 fatty acid eicosapentaenoic acid (EPA) reduces oxidation of ApoB-containing particles in vitro and in patients with hypertriglyceridemia. EPA may produce these effects through a potent antioxidant mechanism, which may facilitate LDL clearance and slow plaque progression. We hypothesize that EPA antioxidant effects may extend to ApoA-containing particles like HDL, potentially preserving certain atheroprotective functions. HDL was isolated from human plasma and incubated at 37 °C in the absence (vehicle) or presence of EPA and/or DHA; 5.0 or 10.0 μM each. Samples were then subjected to copper-induced oxidation (10 μM). HDL oxidation was inhibited similarly by EPA and DHA up to 1 h. EPA (10 μM) maintained significant HDL oxidation inhibition of 89% (0.622 ± 0.066 μM MDA; p < .001) at 4 h, with continued inhibition of 64% at 14 h, vs. vehicle (5.65 ± 0.06 to 2.01 ± 0.10 μM MDA; p < .001). Conversely, DHA (10 μM) antioxidant benefit was lost by 4 h. At a lower concentration (5 μM), EPA antioxidant activity remained at 81% (5.53 ± 0.15 to 1.03 ± 0.10 μM MDA; p < .001) at 6 h, while DHA lost all antioxidant activity by 4 h. The antioxidant activity of EPA was preserved when combined with an equimolar concentration of DHA (5 μM each). EPA pretreatment prevented HDL oxidation in a dose-dependent manner that was preserved over time. These results suggest unique lipophilic and electron stabilization properties for EPA as compared to DHA with respect to inhibition of HDL oxidation. These antioxidant effects of EPA may enhance certain atheroprotective functions for HDL.

 

2.283           Update on the laboratory investigation of dyslipidemias

Ramasamy, I.

Clin. Chim. Acta, 479, 103-125 (2018)

 

The role of the clinical laboratory is evolving to provide more information to clinicians to assess cardiovascular disease (CVD) risk and target therapy more effectively. Current routine methods to measure LDL-cholesterol (LDL-C), the Friedewald calculation, ultracentrifugation, electrophoresis and homogeneous direct methods have established limitations. Studies suggest that LDL and HDL size or particle concentration are alternative methods to predict future CVD risk. At this time there is no consensus role for lipoprotein particle or subclasses in CVD risk assessment. LDL and HDL particle concentration are measured by several methods, namely gradient gel electrophoresis, ultracentrifugation-vertical auto profile, nuclear magnetic resonance and ion mobility. It has been suggested that HDL functional assays may be better predictors of CVD risk. To assess the issue of lipoprotein subclasses/particles and HDL function as potential CVD risk markers robust, simple, validated analytical methods are required. In patients with small dense LDL particles, even a perfect measure of LDL-C will not reflect LDL particle concentration. Non-HDL-C is an alternative measurement and includes VLDL and CM remnant cholesterol and LDL-C. However, apolipoprotein B measurement may more accurately reflect LDL particle numbers. Non-fasting lipid measurements have many practical advantages. Defining thresholds for treatment with new measurements of CVD risk remain a challenge. In families with genetic variants, ApoCIII and lipoprotein (a) may be additional risk factors. Recognition of familial causes of dyslipidemias and diagnosis in childhood will result in early treatment. This review discusses the limitations in current laboratory technologies to predict CVD risk and reviews the evidence for emergent approaches using newer biomarkers in clinical practice.

 

2.284           Isolation and Characterization of Endogenous RNPs from Brain Tissues

Schieweck, R., Ang, F.y., Fritzsche, R. and Kiebler, M.A.

Methods in Mol. Biol., 1649, 419-426 (2018)

 

Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprotein particles that then can be analyzed by mass spectrometry (for proteins composition) and microarray or RNA sequencing technologies (for target mRNAs).

 

2.285           Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell–derived hepatocytes

Schöbel, A., Rösch, K. and Herker, E.

Scientific Reports, 8:3893 (2018)

 

Knowledge of activation and interplay between the hepatitis C virus (HCV) and the hosts’ innate immunity is essential to understanding the establishment of chronic HCV infection. Human hepatoma cell lines, widely used as HCV cell culture system, display numerous metabolic alterations and a defective innate immunity, hindering the detailed study of virus-host interactions. Here, we analysed the suitability of induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) as a physiologically relevant model to study HCV replication in vitro. Density gradients and triglyceride analysis revealed that iHLCs secreted very-low density lipoprotein (VLDL)-like lipoproteins, providing a putative platform for bona fide lipoviroparticles. iHLCs supported the full HCV life cycle, but in contrast to Huh7 and Huh7.5 cells, replication and viral RNA levels decreased continuously. Following HCV infection, interferon-stimulated gene (ISG)-expression significantly increased in iHLCs, whereas induction was almost absent in Huh7/7.5 cells. However, IFNα-stimulation equally induced ISGs in iHLCs and hepatoma cells. JAK-STAT pathway inhibition increased HCV replication in mature iHLCs, but not in Huh7 cells. Additionally, HCV replication levels where higher in STAT2-, but not STAT1-knockdown iHLCs. Our findings support iHLCs as a suitable model for HCV-host interaction regarding a functional innate immunity and lipoprotein synthesis.

 

2.286           Prion-like propagation of β-amyloid aggregates in the absence of APP overexpression

Ruiz-Riquelme, A., Lau, H.H., Stuart, E., Goczi, A.N., Wang, Z., Schmitt-Ulms, G. and Watts, J.C.

Acta Neuropathol. Comm., 6:26 (2018)

 

The amyloid cascade hypothesis posits that the initiating event in Alzheimer’s disease (AD) is the aggregation and deposition of the β-amyloid (Aβ) peptide, which is a proteolytic cleavage product of the amyloid precursor protein (APP). Mounting evidence suggests that the formation and spread of prion-like Aβ aggregates during AD may contribute to disease progression. Inoculation of transgenic mice that overexpress APP with pre-formed Aβ aggregates results in the prion-like induction of cerebral Aβ deposition. To determine whether Aβ deposition can also be induced when physiological APP levels are present in the brain, we inoculated App^NL-F mice, a knock-in model of AD that avoids potential artifacts associated with APP overexpression, with Aβ aggregates derived from the brains of AD patients or transgenic mice. In all cases, induced Aβ deposition was apparent in the corpus callosum, olfactory bulb, and meningeal blood vessels of inoculated mice at 130–150 days post-inoculation, whereas uninoculated and buffer-inoculated animals exhibited minimal or no Aβ deposits at these ages. Interestingly, despite being predominantly composed of protease-resistant Aβ42 aggregates, the induced parenchymal Aβ deposits were largely diffuse and were unreactive to an amyloid-binding dye. These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Aβ deposition. Accordingly, spreading of Aβ deposition may contribute to disease progression in AD patients.

 

 

 

2.287           One‐Way Particle Transport Using Oscillatory Flow in Asymmetric Traps

Lee, J. and Burns, M.A.

Small, 14, 1702724 (2018)

 

One challenge of integrating of passive, microparticles manipulation techniques into multifunctional microfluidic devices is coupling the continuous‐flow format of most systems with the often batch‐type operation of particle separation systems. Here, a passive fluidic technique—one‐way particle transport—that can conduct microparticle operations in a closed fluidic circuit is presented. Exploiting pass/capture interactions between microparticles and asymmetric traps, this technique accomplishes a net displacement of particles in an oscillatory flow field. One‐way particle transport is achieved through four kinds of trap–particle interactions: mechanical capture of the particle, asymmetric interactions between the trap and the particle, physical collision of the particle with an obstacle, and lateral shift of the particle into a particle–trapping stream. The critical dimensions for those four conditions are found by numerically solving analytical mass balance equations formulated using the characteristics of the flow field in periodic obstacle arrays. Visual observation of experimental trap–particle dynamics in low Reynolds number flow (<0.01) confirms the validity of the theoretical predictions. This technique can transport hundreds of microparticles across trap rows in only a few fluid oscillations (<500 ms per oscillation) and separate particles by their size differences.

 

2.288           High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages

Inoue, M., Niki, M., Ozeki, Y., Nagi, S. et al

Scientific Reports, 8:6736 (2018)

 

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

 

2.289           Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin

Mason, R.P., Dawoud, H., Jacob, R.F., Sherratt, S.C.R. and Malinski, T.

Biomedicine Pharmacother., 103, 1231-1237 (2018)

 

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO⁻) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia.

 

2.290           Favouring modulation of circulating lipoproteins and lipid loading capacity by direct antiviral agents grazoprevir/elbasvir or ledipasvir/sofosbuvir treatment against chronic HCV infection

Sun, H-Y., Cheng, P-N., Tseng, C-Y., Tsai, W-J., Chiu, Y-C. and Young, K-C.

Gut, 67, 1342-1350 (2018)

 

Objective Lipid homoeostasis is disturbed in patients with HCV infection. Direct-acting antiviral agent (DAA) treatment eradicates chronic HCV viraemia, but the dynamics of lipid components remain elusive. This study investigates the clinical manifestation and mechanistic relevance of plasma triglyceride (TG), cholesterol (Chol), lipoproteins and apolipoproteins (apos) after DAA treatment.

Design Twenty-four patients with chronic genotype 1 (GT1) HCV treated with elbasvir/grazoprevir or ledipasvir/sofosbuvir for 12 weeks, and followed-up thereafter, were recruited. Their TG, Chol, apoAI and apoB levels were quantified in plasma samples and individually fractionated lipoprotein of various classes. Liver fibrosis was evaluated using the FIB-4 Score. The TG and Chol loading capacities were calculated with normalisation to apoB, which represents per very low density lipoprotein (VLDL) and LDL particle unit

Results DAA treatment achieved a sustained virological response rate of 91.7% and reduced the FIB-4 Score. Relative to the baseline, the plasma TG level was reduced but the Chol level increased gradually. Plasma apoB levels and apoB/apoAI ratio were transiently downregulated as early as the first 4 weeks of treatment. The TG and Chol loading capacities in VLDL were elevated by ~20% during the period of DAA treatment and had steadily increased by 100% at follow-up. Furthermore, the TG-to-Chol ratio in VLDL was increased, while the ratio in LDL was reduced, indicating an efficient catabolism.

Conclusion The DAA treatment of patients with chronic hepatitis C might lead to efficient HCV eradication and hepatic improvement concomitantly evolving with favouring lipoprotein/apo metabolisms.

 

2.291           Dual role of the RNA helicase DDX5 in post-transcriptional regulation of myelin basic protein in oligodendrocytes

Hoch-Kraft, P., White, R., Tenzer, S., Krämer-Albers, E-M., Trotter, J. and Gonsior, C.

1. Cell Science, 131, jcs20470 (2018)

 

In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with Mbp mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with Mbp mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of Mbp. Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.

 

2.292           High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis

Ramakrishnan, S., Gohlke, A., Li, F., Coleman, J., Xu, W., Rothman, J.E. and Pincet, F.

Langmuir, 34(20), 5849-5859 (2018)

 

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

 

2.293           Cryo-EM structure of human mitochondrial trifunctional protein

Liang, K., Li, N., Wang, X., Dai, J., Liu, P., Wang, C., Chen, X-W., Gao, N. and Xiao, J.

PNAS, 115(27), 7039-7044 (2018)

 

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPβ decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.

 

2.294           Mitochondrial maintenance under oxidative stress depends on mitochondrially localised α-OGG1

Lia, D., Reyes, A., de Melo Campos, J.A.T., Piolot, T., Baijer, J., Radicella, J.P. and Campalans, A.

1. Cell Science, 131, jcs213538 (2018)

 

Accumulation of 8-oxoguanine (8-oxoG) in mitochondrial DNA and mitochondrial dysfunction have been observed in cells deficient for the DNA glycosylase OGG1 when exposed to oxidative stress. In human cells, up to eight mRNAs for OGG1 can be generated by alternative splicing and it is still unclear which of them codes for the protein that ensures the repair of 8-oxoG in mitochondria. Here, we show that the α-OGG1 isoform, considered up to now to be exclusively nuclear, has a functional mitochondrial-targeting sequence and is imported into mitochondria. We analyse the sub-mitochondrial localisation of α-OGG1 with unprecedented resolution and show that this DNA glycosylase is associated with DNA in mitochondrial nucleoids. We show that the presence of α-OGG1 inside mitochondria and its enzymatic activity are required to preserve the mitochondrial network in cells exposed to oxidative stress. Altogether, these results unveil a new role of α-OGG1 in the mitochondria and indicate that the same isoform ensures the repair of 8-oxoG in both nuclear and mitochondrial genomes. The activity of α-OGG1 in mitochondria is sufficient for the recovery of organelle function after oxidative stress.

 

2.295           Interfering surface and localized plasmon: Tuning the Wood anomaly for biosensing

Shaimanov, A.N., Orlikovsky, N.A., Khasbushev, E.M., Zverev, A.V., Pishmova, A.A., Sharonov, G.V., Yankovskii, G.M., Rodionov, L.A. and Baryshev, A.V.

Photonics and Nanostructures – Fundamentals and Applications, 32, 1-5 (2018)

 

We demonstrate spectra of slabs of plasmonic 1D nanostructures and show their changes when detecting specific biomolecular binding. The slabs were fabricated by electron-beam lithography, they had sub-millimeter dimensions and allowed us to detect a specific binding of low-density lipoproteins. Optical spectra of the slabs exhibiting a spectrally sharp resonant peak have been analyzed numerically to interpret their features and to define structural parameters governing the quality factor of the resonance. We show a comparison between the sensing performances of the different slabs under study, thus discussing their better designs and experimental geometries.

 

2.296           DNA-induced liquid phase condensation of cGAS activates innate immune signaling

Du, M. and Chen, Z.J.

Science, 361(6403), 704-709 (2018)

 

The binding of DNA to cyclic GMP–AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP–AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling.

 

2.297           Rbfox1 Mediates Cell-type-Specific Splicing in Cortical Interneurons

Warmsley, B., Jaglin, X.H., Favuzzi, E., Khodadadi-Jamayran, A., Rudy, B. and Fishell, G.

Neuron, 100, 846-859 (2018)

 

Cortical interneurons display a remarkable diversity in their morphology, physiological properties, and connectivity. Elucidating the molecular determinants underlying this heterogeneity is essential for understanding interneuron development and function. We discovered that alternative splicing differentially regulates the integration of somatostatin- and parvalbumin-expressing interneurons into nascent cortical circuits through the cell-type-specific tailoring of mRNAs. Specifically, we identified a role for the activity-dependent splicing regulator Rbfox1 in the development of cortical interneuron-subtype-specific efferent connectivity. Our work demonstrates that Rbfox1 mediates largely non-overlapping alternative splicing programs within two distinct but related classes of interneurons.

 

2.298           Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2 

Hangas, A., Aasumets, K., Kekäläinen, N.J., Paloheinä, M., Pohjoismäki, L., Gerhold, J.M. and Goffart, S.

Nucleic Acids Res., 46(18), 9625-9636 (2018)

 

Maintenance of topological homeostasis is vital for gene expression and genome replication in all organisms. Similar to other circular genomes, also mitochondrial DNA (mtDNA) is known to exist in various different topological forms, although their functional significance remains unknown. We report here that both known type II topoisomerases Top2α and Top2β are present in mammalian mitochondria, with especially Top2β regulating the supercoiling state of mtDNA. Loss of Top2β or its inhibition by ciprofloxacin results in accumulation of positively supercoiled mtDNA, followed by cessation of mitochondrial transcription and replication initiation, causing depletion of mtDNA copy number. These mitochondrial effects block both cell proliferation and differentiation, possibly explaining some of the side effects associated with fluoroquinolone antibiotics. Our results show for the first time the importance of topology for maintenance of mtDNA homeostasis and provide novel insight into the mitochondrial effects of fluoroquinolones.

 

2.299           Intermolecular crosslinking of abnormal prion protein is efficiently induced by a primuline-sensitized photoreaction

Teruya, K., Nishizawa, K., Oguma, A., Sakasegawa, Y. and Kitamoto, T.

BBA – General Subjects, 1863, 384-394 (2019)

 

In prion diseases, infectious pathogenic particles that are composed of abnormal prion proteins (PrP^Sc) accumulate in the brain. PrP^Sc is biochemically characterized by its protease-resistance core (PrP^res), but its structural features have not been fully elucidated. Here, we report that primuline, a fluorescent dye with photosensitization activity, dramatically enhances UV-irradiation-induced SDS-resistant PrP^Sc/res oligomer formation that can be detected by immunoblot analysis of prion-infected materials. This oligomer formation occurs specifically with PrP^Sc/res but not with normal prion protein, and it was demonstrated using purified PrP^Sc/res as well as unpurified materials. The oligomer formation proceeded in both primuline-dose- and UV irradiation time-dependent manners. Treatment with urea or formic acid did not break oligomers into monomers. Neither did the presence of aromatic amino acids modify oligomer formation. Analysis with a panel of anti-prion protein antibodies showed that the antibodies against the N-terminal region of PrP^res were less reactive in the dimer than the monomer. These findings suggest that the primuline-sensitized photoreaction enhances intermolecular crosslinking of PrP^Sc/res molecules at a hydrophobic area of the N-terminal region of PrP^res. In the screening of other compounds, photoreactive compounds such as luciferin exhibited a similar but lower activity with respect to oligomer formation than primuline. The enhanced photoreaction with these compounds will be useful for evaluating the structural features of PrP^Sc/res, especially the interactions between PrP^Sc/res molecules.

 

2.300           Cell-associated heparin-like molecules modulate the ability of LDL to regulate PCSK9 uptake

Galvan, A.M. and Chorba, J.S.

1. Lipid Res., 60, 71-84 (2019)

 

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

 

2.301           Membrane trafficking of the bacterial adhesin GspB and the accessory Sec transport machinery

Spencer, C., Bensing, B.A., Mishra, N.N. and Sullam, P.M.

1. Biol. Chem., 294(5), 1502-1515 (2019)

 

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are large, cell wall–anchored adhesins that mediate binding to many host cells and proteins and are associated with bacterial virulence. SRR glycoproteins are exported to the cell surface by the accessory Sec (aSec) system comprising SecA2, SecY2, and 3–5 additional proteins (Asp1 to Asp5) that are required for substrate export. These adhesins typically have a 90-amino acid-long signal peptide containing an elongated N-region and a hydrophobic core. Previous studies of GspB (the SRR adhesin of Streptococcus gordonii) have shown that a glycine-rich motif in its hydrophobic core is essential for selective, aSec-mediated transport. However, the role of this extended N-region in transport is poorly understood. Here, using protein–lipid co-flotation assays and site-directed mutagenesis, we report that the N-region of the GspB signal peptide interacts with anionic lipids through electrostatic forces and that this interaction is necessary for GspB preprotein trafficking to lipid membranes. Moreover, we observed that protein–lipid binding is required for engagement of GspB with SecA2 and for aSec-mediated transport. We further found that SecA2 and Asp1 to Asp3 also localize selectively to liposomes that contain anionic lipids. These findings suggest that the GspB signal peptide electrostatically binds anionic lipids at the cell membrane, where it encounters SecA2. After SecA2 engagement with the signal peptide, Asp1 to Asp3 promote SecA2 engagement with the mature domain, which activates GspB translocation.

2.302           Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup

Heo, P., Ramakrishnan, S., Coleman, J., Rothman, J.E., Fleury, J-B. and Pincet, F.

Small, 15(21), 1900725 (2019)

 

Experimental setups to produce and to monitor model membranes have been successfully used for decades and brought invaluable insights into many areas of biology. However, they all have limitations that prevent the full in vitro mimicking and monitoring of most biological processes. Here, a suspended physiological bilayer‐forming chip is designed from 3D‐printing techniques. This chip can be simultaneously integrated to a confocal microscope and a path‐clamp amplifier. It is composed of poly(dimethylsiloxane) and consists of a ≈100 µm hole, where the horizontal planar bilayer is formed, connecting two open crossed‐channels, which allows for altering of each lipid monolayer separately. The bilayer, formed by the zipping of two lipid leaflets, is free‐standing, horizontal, stable, fluid, solvent‐free, and flat with the 14 types of physiologically relevant lipids, and the bilayer formation process is highly reproducible. Because of the two channels, asymmetric bilayers can be formed by making the two lipid leaflets of different composition. Furthermore, proteins, such as transmembrane, peripheral, and pore‐forming proteins, can be added to the bilayer in controlled orientation and keep their native mobility and activity. These features allow in vitro recapitulation of membrane process close to physiological conditions.

 

2.303           Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer’s Mice with Full-blown Pathology

Schemmert, S., Schartmann, E., Zafiu, C., Kass, B., Hartwig, S., Lehr, S., Bannach, O., Langen, K-J., Shah, N.J., Kutzche, J., Willuweit, A. and Willbold, D.

Mol. Neurobiol., 56, 2211-2223 (2019)

 

Oligomers of the amyloid-β (Aβ) protein are suspected to be responsible for the development and progression of Alzheimer’s disease. Thus, the development of compounds that are able to eliminate already formed toxic Aβ oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aβ oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aβ pathology in old-aged transgenic Alzheimer’s Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aβ oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aβ elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aβ oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer’s disease.

 

2.304           Cellular Trafficking of Amyloid Precursor Protein in Amyloidogenesis Physiological and Pathological Significance

Manucat-Tan, N., Saadipour, K., Wang, Y-J., Bobrovskaya, L. and Zhou, X-F.

Mol. Neurobiol., 56(2), 812-830 (2019)

 

The accumulation of excess intracellular or extracellular amyloid beta (Aβ) is one of the key pathological events in Alzheimer’s disease (AD). Aβ is generated from the cleavage of amyloid precursor protein (APP) by beta secretase-1 (BACE1) and gamma secretase (γ-secretase) within the cells. The endocytic trafficking of APP facilitates amyloidogenesis while at the cell surface, APP is predominantly processed in a non-amyloidogenic manner. Several adaptor proteins bind to both APP and BACE1, regulating their trafficking and recycling along the secretory and endocytic pathways. The phosphorylation of APP at Thr668 and BACE1 at Ser498, also influence their trafficking. Neurotrophins and proneurotrophins also influence APP trafficking through their receptors. In this review, we describe the molecular trafficking pathways of APP and BACE1 that lead to Aβ generation, the involvement of different signaling molecules or adaptor proteins regulating APP and BACE1 subcellular localization. We have also discussed how neurotrophins could modulate amyloidogenesis through their receptors.

 

2.305           New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Ankavay, M., Montpellier, C., Sayed, I.M., Saliou, J-M., Wychowski, C., Saas, L., Duvet, S., Aliouat-Denis, C-M., Farhat, R., de Masson d’Autume, V., Meuleman, P.M Dubuisson, J. and Cocquerel, L.

Scientific Reports, 9:6243 (2019)

 

Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.

 

2.306           The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole

Pike, C.M., Boyer-Andersen, R., Kinch, L.N., Caplan, J.L. and Neunuebel, M.R.

J. Biol. Chem., 294(16), 6405-6415 (2019)

 

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

 

2.307           Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells

AnandBabu, K., Sen, P. and Angayarkanni, N.

PloS One, 14(5), e0216899 (2019)

 

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 μg/mL oxLDL, 500 μM Hcy, 500 nM HCTL, 100 μg/mL AGE, 200 μM H₂O₂ and 200 μM H₂O₂ with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-κB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-κB activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.

 

2.308           Chimaeric Rift Valley Fever Virus‐Like Particle Vaccine Candidate Production in Nicotiana benthamiana

Mbewana, S., Meyers, A.E. and Rybicki, E.P.

Biotech. J., 14(4), 1800238 (2019)

 

Rift Valley fever virus (RVFV) is an emerging mosquito‐borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live‐attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus‐like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens‐mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail‐encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg⁻¹ fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne‐HA particles of 49–60 nm which are immunogenic, eliciting Gn‐specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne‐HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

 

2.309           The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins

Thierer, J.H., Ekker, S.C. and Farber, S.A.

Nature Communications, 10:3426 (2019)

 

Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect images of atherogenic lipoprotein localization in an intact organism. We report multiple extravascular lipoprotein localization patterns, as well as identify Pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.

 

2.310           A programmable DNA-origami platform for studying lipid transfer between bilayers

Bian, X., Zhang, Z., Xiong, Q., De Camilli, P. and Lin, C.

Nature Chem. Biol., 15, 830-837 (2019)

 

Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other.

 

2.311           The role of surface chemistry in serum protein corona-mediated cellular delivery and gene silencing with lipid nanoparticles

Chen, D., Ganesh, S., Wang, W. and Amiiji, M.

Nanoscale, 11, 8760-8775 (2019)

 

Delivery of genetic medicines, such as small interfering RNA (siRNA), by lipid nanoparticles (LNPs) is a promising approach towards the treatment of diseases, such as solid tumors. However, in vitro and in vivo nanoparticle delivery efficiency is influenced by the formation of a protein corona in biological media. In this study, we have formulated four types of EnCore nanoparticles (F1 to F4) with a similar composition, but different polyethylene glycol (PEG) conjugated lipid chain lengths (carbon 14 vs. carbon 18) and molar ratios (6% vs. 3%). These LNPs showed dramatic differences in cellular delivery and transfection in hepatocellular carcinoma (HepG2) cells in the absence and presence of fetal bovine serum (FBS). The presence of proteins inhibited the cellular uptake of C18 (3%) nanoparticles, while it facilitated the cellular uptake of C14 nanoparticles. Among the adsorbed proteins from FBS, apolipoprotein E, but not apolipoprotein A1, affected the cellular uptake of the carbon 14 LNPs. Additionally, surface PEG was one of the determinants for the protein corona amount and composition. Finally, different serum to LNP volume ratios resulted in different protein enrichment patterns. Overall, the results showed a correlation between surface chemistry of LNPs and the protein corona composition suggesting a potential use for targeted delivery.

 

2.312           Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway

Igel-Egalon, A., Laferriere, F., Moudjou, M., Bohl, J., Mezache, M., Knäpple, T., Herzog, L., Reine, F., Jas-Duval, C., Doumic, M., Rezaei, h. and Beringue, V.

Communications Biol., 2:363 (2019)

 

The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.

 

2.313           Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans

Wagner, A., Hofmeister, O., Rolland, S.G., maiser, A., Aasumets, K., Schmitt, S., Schorpp, K., Feuchtinger, A., Hadian, K., Scheneider, S., Zischka, H., Leonhardt, H., Conradt, B., Gerhold, J.M. and Wolf, A.

1. Cell Sci., 132, jcs223891 (2019)

 

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N³-methylcytosine (m³C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N¹-methyladenosine (m¹A) in tRNA or formation of 5-formyl cytosine (f⁵C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPR^mt) in C. elegans.

 

2.314           The role of apolipoprotein- and vitronectin-enriched protein corona on lipid nanoparticles for in vivo targeted delivery and transfection of oligonucleotides in murine tumor models

Chen, D., Parayath, N., Ganesh, S., Wnag, W. and Amiji, M.

Nanoscale, 11, 18806-18824 (2019)

 

The application of lipid-based nanoparticle (LNP) delivery systems remains a popular strategy for the systemic delivery of gene therapies to specific disease targets, including solid tumors. It is now well acknowledged that upon systemic administration, biomolecules from blood will adsorb onto nanoparticles’ surfaces, forming a “protein corona”, affording nanoparticles a “biological identity” on top of their “synthetic identity”. Detailed analysis of nanoparticle protein corona is gradually revealing the “missing link” between nanoparticle chemical properties and the biological identity. Nevertheless, the discovery of nanoparticle protein corona's impact on tumor delivery is limited. In this study, we demonstrate that protein corona can be manipulated by formulation composition and particle surface charge changes, and a single lipid switch could switch the nanoparticle protein corona profile. The protein corona composition differences had a profound impact on cell transfection, in vivo biodistribution as well as tumor-specific delivery efficiency. Nanoparticles with apolipoprotein-rich corona showed better delivery to hepatocellular carcinoma (HepG2) as compared to those with vitronectin-rich corona. In addition, we found that, the PEG conjugated lipid chain length and PEG amount in LNPs were key factors to consider in successful RNA interference therapy for solid tumors.

 

2.315           Elevated Lipoprotein(a) Levels Lower ABCA1 Cholesterol Efflux Capacity

Tavori, H., Fenton, A.M., Plubell, D.L., Rosario, S., Yerkes, E., Gasik, R., Miles, J., Bergstrom, P., Minnier, J., Fazio, S. and Pamir, N.

1. Clin. Endocrinol. Metab., 104(10), 4793-4803 (2019)

 

Context

Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a).

Objective

To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux.

Methods

Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels.

Results

Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (−30.6%, P = 0.002 vs −24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = −0.433, P = 0.0007 vs r = −0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R² = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R² = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux.

Conclusion

Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.

 

2.316           Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

Ziveri, J., Chhuon, C., Jamet, A., Rytter, h., Prigent, G., tros, F., Barel, M., Coureuil, M., Lays, C., Henry, T., Keep, N.H., Guerrera, I.C. and Charbit, A.

Mol. Cell. Proteomics, 18, 2418-2432 (2019)

 

The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774–1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.

 

2.317           Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

Abskharon, R., Wang, F., Wohlkonig, A., Ruan, J., Soror, s., Giachin, G., Pardon, E., Zou, W., Legname, G., Ma, J. and Steyaert, J.

PloS Pathogens, 15(12), e1008139 (2019)

 

Prion or PrP^Sc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrP^Sc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrP^Sc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

 

2.318           A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles

Sarkar, S.K., Foo, A.C.Y., Matyas, A., Asikhia, I., Kosenko, T., Goto, N.K., Vergara-jaque, A. and Lagace, T.A.

1. Biol. Chem., 295(8), 2285-2298 (2020)

 

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

 

2.319           Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro

Sherratt, S.C.R., Juliano, R.A. and Mason, R.P.

BBA-Biomembranes, 1862, 183254 (2020)

 

Background

Oxidation of small dense low-density lipoprotein (sdLDL) and membranes is causally related to atherosclerosis. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA, 20:5, ω-3) significantly reduced oxidized LDL in patients with hypertriglyceridemia by unknown mechanisms. We compared EPA effects to related FAs of varying chain length and unsaturation on oxidation of sdLDL and model membranes, and on cholesterol crystal domains. We compared EPA to the FAs: stearic (SA, 18:0), oleic (OA, 18:1, ω-9), linoleic (LA, 18:2, ω-6), alpha-linolenic (ALA, 18:3, ω-3), eicosanoic (EA, 20:0), eicosatrienoic (ETE, 20:3, ω-3), arachidonic (AA, 20:4, ω-6), docosapentaenoic (DPA, 22:5, ω-3), and docosahexaenoic (DHA, 22:6, ω-3).

Methods

Human sdLDL or model membranes of cholesterol and 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine [18:2(cis)PC or DLPC] were preincubated with FAs followed by copper-induced oxidation. Malondialdehyde (MDA) or lipid hydroperoxides (LOOH) levels measured oxidation; small-angle X-ray diffraction assessed cholesterol domain formation.

Results

After 40 min, EPA reduced MDA levels 70% compared to vehicle (p < 0.001). Lesser inhibition was observed with DHA, DPA, ETE, and ALA (33%, 34%, 32%, and 16%, respectively; all p < 0.001 versus vehicle). Similar relative FA effects were observed in model membranes where EPA more substantially inhibited cholesterol crystal domain formation.

Conclusion

We observed relationships between hydrocarbon length and unsaturation with antioxidant activity and membrane cholesterol domain formation. EPA had the most favorable molecular structure, likely contributing to membrane stability, improved lipoprotein clearance, and reduced inflammation.

 

2.320           A muscle-specific calpain, CAPN3, forms a homotrimer

Hata, S., Doi, N., Shinkai-Ouchi, F. and Ono, Y.

BBA-Proteins and Proteomics, 1868, 140411 (2020)

 

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.

 

2.321           Sciadonic acid derived from pine nuts as a food component to reduce plasma triglycerides by inhibiting the rat hepatic Δ9-desaturase

Pedrono, F., Boulier-Monthean, N., Boissel, F., Ossemond, J., Viel, R., Fautrel, A., Marchix, J. and Dupont, D.

Scientific Reports, 10:6223 (2020)

 

Sciadonic acid (Scia) is a Δ5-olefinic fatty acid that is particularly abundant in edible pine seeds and that exhibits an unusual polymethylene-interrupted structure. Earlier studies suggested that Scia inhibited the in vitro expression and activity of the Stearoyl-CoA Desaturase 1 (SCD1), the hepatic Δ9-desaturase involved in the formation of mono-unsaturated fatty acids. To confirm this hypothesis, rats were given 10% Scia in diets balanced out with n-6 and n-3 fatty acids. In those animals receiving the Scia supplement, monoene synthesis in the liver was reduced, which was partly attributed to the inhibition of SCD1 expression. As a consequence, the presence of Scia induced a 50% decrease in triglycerides in blood plasma due to a reduced level of VLDL-secreted triglycerides from the liver. In non-fasting conditions, results showed that Scia-induced inhibition of SCD1 led to a decrease in the proportions of 16:1n-7 and 18:1n-7 in the liver without impacting on the level of 18:1n-9, suggesting that only triglycerides with neosynthesized monoenes are marked out for release. In conclusion, this in vivo study confirms that Scia highly inhibits SCD1 expression and activity. The work was performed on normo-triglyceride rats over six weeks, suggesting promising effects on hyper-triglyceridemic models.

 

2.322           Prion protein post-translational modifications modulate heparan sulfate binding and limit aggregate size in prion disease

Callender, J.A., Sevillano, A.M., Soldau, K., Kurt, T.D., Schumann, T., Pizzo, D.P., Altmeppen, H., Glatzel, M., Esko, J.D. and Sigurdson, C.J.

Neurobiol. of Disease, 142, 104955 (2020)

 

Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. These plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate (HS) similar to other predominantly GPI-anchored prions. Collectively, these results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform, with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Since PrP^C is highly glycosylated, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits in the brain as well as the rapid conversion commonly observed in human and animal prion disease.

 

2.323           Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

Melville, D. B., Studer, S. and Schekman, R.

1. Biol. Chem., 295(25), 8401-8412 (2020)

 

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

 

2.324           pH-responsive Frame-Guided Assembly with hydrophobicity controllable peptide as leading hydrophobic groups

Wang, C., Zhang, Y., Shao, Y., Tian, X., Piao, J., Dong, Y. and Liu, D.

Giant, 1, 100006 (2020)

 

Frame-Guided Assembly (FGA) strategy has been recently reported to prepare vesicles with customized shapes and sizes. However, the effects of the interaction between leading hydrophobic groups (LHGs) and amphiphiles on the thermodynamic and kinetic control of the FGA process haven't been fully understood. In this work, we employed the pH low-insertion peptide (pHLIP) as the LHGs because its interaction with lipids could be finely tuned by pH and investigated the mechanism of FGA in detail. Our study demonstrated the peptide frames could successfully guide the assembly of lipids to form hetero-liposomes below the pH transition point owing to the strong peptide and lipids interaction. The pH-dependent kinetic controlled FGA process was proved and factors affecting the FGA process were also investigated systematically. We believe this pH-responsive FGA strategy improved our knowledge on the mechanism of the FGA and provide inspiration in understanding the sophisticated assembly behavior in life.

 

2.325           Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease

Vanni, I., Pirisinu, L., Acevedo-Morantes, C., Kamali-Jamil, R., Rathod, V. et al

Brain, 143(5), 1512-1524 (2020)

 

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrP^Sc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrP^Sc aggregates, whose protease-resistant core (PrP^res) encompasses the whole C-terminus of PrP. In contrast, PrP^Sc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrP^Sc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (10^9.3 ID₅₀ U/g) and is resistant to treatment with proteinase K (10^9.0 ID₅₀ U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrP^res in the different fractions, alongside the morphological characteristics of purified PrP^res aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrP^res, although the yield of PrP^res was low. We found that this low yield depended on the low density/small size of GSS-A117V PrP^res, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrP^res, with infectivity levels that directly correlated with the PrP^res amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrP^resparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrP^res, spanning residues ∼90–150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrP^Sc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.

 

2.326           Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

Ellison, C.J., Kukulski, W., Boyle, K.B., Munro, S. and Randow, F.

Current Biol., 30, 2974-2983 (2020)

 

Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2, 3, 4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7, 8, 9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.

 

2.327           Exploring the effects of carrier oil type on in vitro bioavailability of β-carotene: A cell culture study of carotenoid-enriched nanoemulsions

Xia, Z., Han, Y., Du, H., McClements, D.J., Tang, Z. and Xiao, H.

LWT-Food Sci. Technol., 134, 110224 (2020)

 

β-carotene was encapsulated in either olive oil or flaxseed oil-in-water nanoemulsions, and it's in vitro bioaccessibility and bioavailability were determined in a simulated gastrointestinal digestion model (mouth, stomach and small intestine) combined with a Caco-2 cell monolayer model. Nanoemulsions fabricated from both types of oils significantly increased the in vitro bioaccessibility and bioavailability of β-carotene. Olive oil, however, was digested more efficiently, which generated more free fatty acids capable of forming mixed micelles. Furthermore, the mixed micelles stimulated the formation of lipoprotein particles (chylomicrons and very low-density lipoproteins), which are the transcellular carriers of β-carotene in the intestinal epithelium, leading to an increase in bioavailability. Interestingly, olive oil led to the formation of larger chylomicrons than flaxseed oil, suggesting that fatty acid type impacts the nature of the lipoprotein particles formed after lipid digestion.

 

2.328           A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP

Munoz-Montesino, C., larkem, D., Barbereau, C., Igel-Egalon, A., Truchet, S., Jacquet, E., Nhiri, N., Moudjou, M., Sizun, C., Rezaei, H., Beringue, V.and Dron, M.

J. Biol. Chem., 295(41), 14025-14039 (2020)

 

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet–rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)–resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190–196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

 

2.329           Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy

Laferriere, F., He, X., Zinghirino, F., Doudnikoff, E., Faggiani, E., Meissner, W.G., Bezard, E., De Giorgi, F. and Ichas, F.

Cells, 9:2371 (2020)

 

The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar α-synuclein (α-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson’s disease (PD) where the pathological α-syn aggregates are almost exclusively neuronal, the α-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of α-syn, yet pathogenic amyloid α-syn seeds require significant amounts of α-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human α-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated α-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated α-syn monomers/oligomers and not to the appearance of the distinctive fibrillar α-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-αSyn mice we observed a variable association of S129-phosphorylated α-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-α-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with α-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of α-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between α-syn aggregation and the development of a clinical phenotype in these transgenic animals.

 

2.330           Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

Wickham, S.F.J., Auer, A., Min, J., Ponnuswamy, N., Wooehrstein, J.B., Schueder, F. et al

Nature Communications, 11:5768 (2020)

 

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.

 

2.331           RCC1L (WBSCR16) isoforms coordinate mitochondrial ribosome assembly through their interaction with GTPases

Reyes, A., Favia, P., Vidoni, S., Petruzzella, V. and Zeviani, M.

PloS Genetics, 16(7), e1008923 (2020)

 

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1L^V1, RCC1L^V2 and RCC1L^V3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1L^V1 and RCC1L^V3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1L^V2. Overexpression and silencing of RCC1L^V1 or RCC1L^V3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.

 

2.332           Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression

Pareek, G. and Pallanck, L.J.

PloS Genetics, 16(10), e1009118 (2020)

 

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2.

 

2.333           Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates

Bourdenx, M., Nioche, A., Dovero, S., Arotcarena, M.L., Camus, S., Porras, G. et al

Sci. Adv., 6:eaa9165 (2020)

 

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)–rich protein aggregates [termed “Lewy bodies” (LBs)], is a well-established characteristic of Parkinson’s disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning–based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

 

2.334           The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis)

Sirichaiwetchakoon, K., Lowe, G.M. and Eumkeb, G.

BioMed Res. Int., 2020:4183643 (2020)

 

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3- ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu2+), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH (P < 0:05; IC50PIT = 245:85 ± 15:83 and CST = 315:41 ± 24:18 μg/ml) and peroxynitrite scavenging assays. PIT at 75 μg/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0:05; IC50PIT = 30:47 ± 2:20 and CST = 21:59 ± 0:67 μg/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress

 

2.335           Optimizing the method of plasma lipoprotein isolation for elucidating their differential association to proprotein convertase subtilisin/kexin 9 (PCSK9)

Canclini, L., Malvandi, A.M., Uboldi, P., Zampoleri, V., Bellosta, S., Bargetti, A., Grigore, l., Zambon, A. and Catapano, A.L.

Atherosclerosis, 315, e161 (2020)

 

Background and Aims: PCSK9 is a secretory protease that has emerged as a target to regulate lipoprotein metabolism. Antibodies blocking PCSK9 reduce LDL-C levels through the prevention of PCSK9 binding to the LDL receptor. The existence of PCSK9-lipoprotein complexes is reported, but data in literature are discordant. This project aims at studying the existence of this interaction and its possible biological consequences. To reach this goal, we aimed at setting the optimal condition in the lipoprotein isolation method that helps build up the capacity for future clinical studies.

Methods: Fresh sera were collected from 60 healthy individuals to isolate the lipoprotein fraction using different methods including precipitation with phosphotungstic acid, fast protein liquid chromatography (FPLC), ultracentrifugation using KBr or iodixanol gradient (IGr), as a nonionic hydrophilic compound that would not interfere with molecular associations. The resulting fractions were analyzed to detect PCSK9 with ELISA and LP(a), ApoB, ApoA1, and Cholesterol using clinical-grade turbidimetry assays.

Results: In the precipitation-mediated assay, around 60% of PCSK9 was found in ApoB fraction. In the FPLC, 10±0.5% of recovered PCSK9 was detected in the LDL fraction. No PCSK9 was detectable in lipoprotein fraction isolated by ultracentrifugation in the presence of KBr, whereas 23±8% of PCSK9 was found in LDL fraction isolated with the iodixanol gradient.

Conclusions: Based on our observations, it appears that the association of PCSK9 and LDL is sensitive to salt concentrations and changes with the isolation method. No binding to other lipoproteins was detected. More studies are requested to define the type of interaction.

 

2.336           Adiponectin and related C1q/TNF-related proteins bind selectively to anionic phospholipids and sphingolipids

Ye, J.J., Bian, X., Lim, J. and Medzhitov, R.

PNAS, 117(229), 17381-17388 (2020)

 

Adiponectin (Acrp30) is an adipokine associated with protection from cardiovascular disease, insulin resistance, and inflammation. Although its effects are conventionally attributed to binding Adipor1/2 and T-cadherin, its abundance in circulation, role in ceramide metabolism, and homology to C1q suggest an overlooked role as a lipid-binding protein, possibly generalizable to other C1q/TNF-related proteins (CTRPs) and C1q family members. To investigate this, adiponectin, representative family members, and variants were expressed in Expi293 cells and tested for binding to lipids in liposomes using density centrifugation. Binding to physiological lipids were also analyzed using gradient ultracentrifugation, liquid chromatography-mass spectrometry, and shotgun lipidomics. Interestingly, adiponectin selectively bound several anionic phospholipids and sphingolipids, including phosphatidylserine, ceramide-1-phosphate, glucosylceramide, and sulfatide, via the C1q domain in an oligomerization-dependent fashion. Binding to lipids was observed in liposomes, low-density lipoproteins, cell membranes, and plasma. Other CTRPs and C1q family members (Cbln1, CTRP1, CTRP5, and CTRP13) also bound similar lipids. These findings suggest that adiponectin and CTRPs function not only as hormones, but also as lipid opsonins, as may other C1q family proteins.

 

2.337           Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice

Degenhardt, K., Wagner, J., Skodras, A., Candlish, M. et al

PNAS, 117(38), 23925-23931 (2020)

 

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

 

2.338           Range of SHH signaling in adrenal gland is limited by membrane contact to cells with primary cilia

Mateska, I., Nanda, K., Dye, N.A., Alexaki, V.I. and Eaton, S.

1. Cell Biol., 219(12), e201910087 (2020)

 

The signaling protein Sonic Hedgehog (SHH) is crucial for the development and function of many vertebrate tissues. It remains largely unclear, however, what defines the range and specificity of pathway activation. The adrenal gland represents a useful model to address this question, where the SHH pathway is activated in a very specific subset of cells lying near the SHH-producing cells, even though there is an abundance of lipoproteins that would allow SHH to travel and signal long-range. We determine that, whereas adrenal cells can secrete SHH on lipoproteins, this form of SHH is inactive due to the presence of cosecreted inhibitors, potentially explaining the absence of long-range signaling. Instead, we find that SHH-producing cells signal at short range via membrane-bound SHH, only to receiving cells with primary cilia. Finally, our data from NCI-H295R adrenocortical carcinoma cells suggest that adrenocortical tumors may evade these regulatory control mechanisms by acquiring the ability to activate SHH target genes in response to TGF-β.

 

2.339           EIF3H Orchestrates Hippo Pathway–Mediated Oncogenesis via Catalytic Control of YAP Stability

Zhou, Z., Zhou, H., Ponzoni, L., Luo, A., He, M., Huang, Y., Guan, K-L-. Bahar, I., Liu, Z. and Wan, Y.

Cancer Res., 80, 2550-2563 (2020)

 

EIF3H is presumed to be a critical translational initiation factor. Here, our unbiased screening for tumor invasion factors has identified an unexpected role for EIF3H as a deubiquitylating enzyme that dictates breast tumor invasion and metastasis by modulating the Hippo–YAP pathway. EIF3H catalyzed YAP for deubiquitylation, resulting in its stabilization. Structure-based molecular modeling and simulations coupled with biochemical characterization unveiled a unique catalytic mechanism for EIF3H in dissociating polyubiquitin chains from YAP through a catalytic triad consisting of Asp90, Asp91, and Gln121. Trp119 and Tyr 140 on EIF3H directly interacted with the N-terminal region of YAP1, facilitating complex formation of EIF3H and YAP1 for YAP1 deubiquitylation. Stabilization of YAP via elevated EIF3H promoted tumor invasion and metastasis. Interference of EIF3H-mediated YAP deubiquitylation blocked YAP-induced tumor progression and metastasis in breast cancer models. These findings point to a critical role for YAP regulation by EIF3H in tumor invasion and metastasis.

 

2.340           Association of circulating levels of total and protein-bound sphingosine 1-phosphate with osteoporotic fracture

Song, H.E., Lee, S.H., Kim, S.J., Kim, B-J., Yoo, H.J. and Koh, J-M.

1. Investig. Med., 68, 1295-1299 (2020)

 

The biological activity and effects of circulating sphingosine 1-phosphate (S1P) might be dependent on the carrier protein. Although S1P is known to be a biomarker for osteoporotic fracture (OF), its role according to its carrier protein (high-density lipoprotein (HDL), low-density lipoprotein (LDL), or albumin) has not yet been studied. We measured the protein-bound S1P levels and bone mineral density (BMD) in 58 postmenopausal women with OF and 58 age-matched and body mass index–matched postmenopausal women without OF. Albumin-bound S1P was the most abundant. Before adjustment, women with OF had higher total S1P (p=0.046) and albumin-bound S1P (p=0.026) levels than those without OF, but there was no difference in the levels of HDL-bound or LDL-bound S1P. After adjustment for confounders including BMD, women with OF had only higher levels of total S1P than those without OF (p=0.047). Before adjustment, the OR for OF was higher in subjects in the highest quartile for total S1P (OR 5.36, 95% CI 1.22 to 23.63) or albumin-bound S1P (OR 4.48, 95% CI 1.22 to 16.42). After adjustment for confounders including BMD, statistical significance persisted only for total S1P (OR 2.23, 95% CI 1.12 to 4.81). These findings suggest that the positive association of S1P with OF is mainly due to level of total plasma S1P and not due to the differing contributions from specific carrier protein-bound fractions.

 

2.341           Unveiling the pitfalls of the protein corona of polymeric drug nanocarriers

Berrecoso, G., Crecente-Campo, J. and Alonso, M.J.

Drug Delivery and Transl. Res., 10, 730-750 (2020)

 

The protein corona is a natural protein layer spontaneously formed around nanomaterials when exposed to biological media. This layer can alter the nanosystems’ biological performance, particularly their tissue accumulation, cellular uptake, clearance by the immune system, toxicity, and even the release profile of their payloads. Hence, the characterization of this protein layer has become a critical step when developing a new nanomedicine. The modification of the nanosystem fate by the protein corona, systematically ignored in the vast majority of the nanotechnology-based research, may have contributed to the low in vitro/in vivo correlation. Actually, the protein corona of polymeric nanosystems has been scarcely studied in the literature, and most studies have been focused instead on metallic nanoparticles and liposomes. In this review, we analyzed the influence of the physicochemical properties and composition of the polymeric nanosystems on the protein layer deposited around them. In addition, we present some recommendations on how to perform the protein corona studies of polymeric nanoparticles, which, hopefully, will contribute to obtain more reliable and reproducible data in the future.

 

2.342           High-Density Lipoproteins Are the Main Carriers of PCSK9 in the Circulation

S.A. Burnap and M. Mayr et. al.

1. Am. Coll. Cardial., 75(12), 2667-2676 (2020)

 

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that regulates circulating low-density lipoprotein (LDL) through the hepatic LDL receptor degradation pathway, is the latest therapeutic target to further lower cholesterol in patients on maximal statin therapy (1). Circulating PCSK9 has been shown to bind to LDL and lipoprotein(a) (Lp[a]). The latter is an LDL particle carrying apolipoprotein(a) as an additional protein component (2,3). Furthermore, the multimeric state of PCSK9 is thought to be influenced by lipoproteins, including high-density lipoprotein (HDL), regulating the LDL receptor–degrading capabilities of PCSK9 (4). However, the presence of PCSK9 on other lipoprotein particles is less well established, in particular in humans.

The potential associations of PCSK9 with different human lipoproteins was first determined by immunocapture, as previously described (5). Alirocumab, a human monoclonal antibody to PCSK9, was coated as a capture antibody to measure plasma PCSK9 in 20 healthy volunteers. Antibodies specific for apolipoprotein(a), apolipoprotein B (ApoB), and apolipoprotein A1 were then used to interrogate PCSK9-lipoprotein associations with Lp(a), ApoB-containing lipoproteins, and HDL, respectively. Measurement of lipoprotein-associated PCSK9 suggested a predominant association of PCSK9 with HDL (Figure 1A). For validation, plasma was subject to an anti-ApoB immunoprecipitation (Sun Diagnostics, New Gloucester, Maine). Successful removal of ApoB-carrying lipoproteins, including Lp(a), as confirmed by targeted mass spectrometry, led to only a <20% removal of PCSK9 as measured by enzyme-linked immunosorbent assay (DY3888, R&D Systems, Minneapolis, Minnesota; data not shown). In contrast, HDL removal using an HDL immunodepletion column (Genway Biotech, San Diego, California) resulted in a >90% removal of PCSK9 from fasting plasma (data not shown).

 

2.343           Pharmacologic Suppression of B7-H4 Glycosylation Restores Antitumor Immunity in Immune-Cold Breast Cancers

Sosng, X., Zhou, Z., Li, H., Xue, Y., Lu, X., Baha, I., Keep, O., Hung, M-C., Kroemer, G. and Wan, Y.

CancerDiscov., 10(12), 1872-1893 (2020)

 

Despite widespread utilization of immunotherapy, treating immune-cold tumors has proved to be a challenge. Here, we report that expression of the immune checkpoint molecule B7-H4 is prevalent among immune-cold triple-negative breast cancers (TNBC), where its expression inversely correlates with that of PD-L1. Glycosylation of B7-H4 interferes with its interaction/ubiquitination by AMFR, resulting in B7-H4 stabilization. B7-H4 expression inhibits doxorubicin-induced cell death through the suppression of eIF2α phosphorylation required for calreticulin exposure vis-à-vis the cancer cells. NGI-1, which inhibits B7-H4 glycosylation causing its ubiquitination and subsequent degradation, improves the immunogenic properties of cancer cells treated with doxorubicin, enhancing their phagocytosis by dendritic cells and their capacity to elicit CD8⁺ IFNγ-producing T-cell responses. In preclinical models of TNBC, a triple combination of NGI-1, camsirubicin (a noncardiotoxic doxorubicin analogue) and PD-L1 blockade was effective in reducing tumor growth. Collectively, our findings uncover a strategy for targeting the immunosuppressive molecule B7-H4.

 

2.344           Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion

Matoba, K., Kotani, T., Tsutsumi, A., Tsuji, T., Mori, T., Noshiro, D., Sugita, Y., Nomura, n., Iwata, S., Ohsumi, Y., Fujimoto, T., Nakatogawa, H., Kikkawa, M. and Noda, N.N.

Nature Struct. Mol. Biol., 27, 1185-1193 (2020)

 

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

 

2.345           A critical review: Recent advances in “digital” biomolecule detection with single copy sensitivity

Liu, H. and Lei, Y.

Biosensors and Bioelectronics, 177, 112901 (2021)

 

Detection of a single biomolecule, ranging from nucleic acids, proteins, viruses to bacteria, is of paramount importance in various fields including biology, environment, food and agriculture industry, public health, and medicine. With the understanding of the biological functions of these biomolecules (or bioparticles) and their impacts on public health, environmental pollution, and food safety, advanced detection techniques are unprecedentedly demanded for their early and/or sensitive detection. In this critical review, a series of elegant research about digital detection of biomolecules with potential single copy sensitivity is reviewed and summarized with the focus on the design principle and the innovation of how to accomplish the “digital” detection concept. Starting with a brief introduction on the importance of digital detection, recent advances in “digital” biomolecule detection with single copy sensitivity are grouped and discussed based on the difference of signal reporting systems, including surrogate signal development for “digital” detection, direct visualization for “digital” detection, and nucleic acid amplification enabled “digital” detection. Interdisciplinary combination and integration of different cutting-edge techniques are also discussed with details. The review is closed with the conclusion and future trends.

 

2.346           Expanding the codes: The development of density-encoded hydrogel microcarriers for suspension arrays

Hou, M., Shi, L., Zhou, Y., Wang, J., Jiang, J., Jiang, j. and He, J.

Biosensors and Bioelectronics, 181, 113133 (2021)

 

Although suspension array technology (SAT), which uses encoded microspheres, provides high-quality results with versatile applicability for information-intensive bioanalytic applications, current encoding strategies limit the number of codes that can be distinguished. In this paper, we introduce density-encoded hydrogel microcarriers (DMs), which employ the intrinsic density property of biomaterials as a high-capacity coding dimension. Two hydrogel monomers were employed at different ratios to synthesize microgels with distinctive densities. DMs not only can be simultaneously decoded and separated using density gradient centrifugation, but also are compatible with flow cytometry detection. The size and color of DMs have been used as extra coding parameters, to construct an 8 × 2 × 4 (density × size × color) three-dimensionally encoded hydrogel microcarrier library. With aptamer-functionalized DMs (ADMs), we developed a 4-plex protein quantification method for the label-free detection of plasma biomarkers with sub-nanomolar detection limits and good linearities. Moreover, ADMs can be used for label-free naked-eye detection of tumor-derived exosomes. We believe that the simplicity and functionality of DMs will advance the field of suspension arrays and inspire the development of DM-based diagnostic applications.

 

2.347           Lipoprotein compartmentalisation as a regulator of PCSK9 activity

Burnap, S.A. and Mayr, M.

1. Mol. Cell. Cardiol., 155, 21-24 (2021)

 

Lipoprotein compartmentalisation and PCSK9-mediated LDLR degradation. PCSK9 binds to the extracellular domain of the LDLR to promote its degradation through the lysosomal compartment. PCSK9 exists in a free and lipoprotein-bound form in the circulation. PCSK9 association with LDL is thought to sequester PCSK9 and inhibit PCSK9-mediated LDLR degradation. The functional consequence of the interaction between PCSK9 and HDL is currently unknown. PCSK9 probably shuttles between a lipoprotein-bound pool of PCSK9 and circulating free-PCSK9. This lipoprotein compartmentalisation may impact on its LDLR degrading activity.

 

 

2.348           A Comparative Analysis of the Lipoprotein(a) and Low-Density Lipoprotein Proteomic Profiles Combining Mass Spectrometry and Mendelian Randomization

Bourgeois, R., Girard, A., Perrot, N., Guertin, J., Mirchell, P.L., Couture, Cc. et al

CJC Open, 3, 450-459 (2021)

 

Background

Lipoprotein(a) (Lp[a]), which consists of a low-density lipoprotein (LDL) bound to apolipoprotein(a), is one of the strongest genetic risk factors for atherosclerotic cardiovascular diseases. Few studies have performed hypothesis-free direct comparisons of the Lp(a) and the LDL proteomes. Our objectives were to compare the Lp(a) and the LDL proteomic profiles and to evaluate the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile.

Methods

We performed a label-free analysis of the Lp(a) and LDL proteomic profiles of healthy volunteers in a discovery (n = 6) and a replication (n = 9) phase. We performed inverse variance weighted Mendelian randomization to document the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile of participants of the INTERVAL study.

Results

We identified 15 proteins that were more abundant on Lp(a) compared with LDL (serping1, pi16, itih1, itih2, itih3, pon1, podxl, cd44, cp, ptprg, vtn, pcsk9, igfals, vcam1, and ttr). We found no proteins that were more abundant on LDL compared with Lp(a). After correction for multiple testing, lifelong exposure to elevated LDL cholesterol levels was associated with the variation of 18 plasma proteins whereas Lp(a) did not appear to influence the plasma proteome.

Conclusions

Results of this study highlight marked differences in the proteome of Lp(a) and LDL as well as in the effect of lifelong exposure to elevated LDL cholesterol or Lp(a) on the plasma proteomic profile.

 

 

2.349           TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

Aasumets, K., Basikhina, Y., Pohjoismäki, J., Goffart, S. and Gerhold, J.

Biochem. Biophys. Reports, 28, 101142 (2021)

 

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.

 

2.350           The role of the Cer1 transposon in horizontal transfer of transgenerational memory

Moore, R.S., Kaletsky, R.S., Lesnik, C., Parsons, L.R., Gital, Z. and Murphy, C.T.

Cell, 184, 4697-4712 (2021)

 

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. C. elegans protects itself from pathogens by “reading” bacterial small RNAs, using this information to both induce avoidance and transmit memories for four generations. Here, we found that memories can be transferred from either lysed animals or from conditioned media to naive animals via Cer1 retrotransposon-encoded virus-like particles. Moreover, Cer1 functions internally at the step of transmission of information from the germline to neurons and is required for learned avoidance. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small-RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

 

2.351           An evaluation of different methods to study the association of proprotein convertase subtilisin-kexin type 9 to lipoproteins.

Canclini, L., Malvandi, A.M., Uboldi, P., Zampoleri, V., Baragetti, A., Grigore, L., and Catapano, A.L.

Atherosclertosis, 331, e139 (2021)

 

Background and Aims: Proprotein convertase subtilisin-kexin type 9 (PCSK9) enhances the degradation of the hepatic low-density lipoprotein (LDL) receptor, increasing LDL-cholesterol levels in plasma. PCSK9 has been reported to associate with lipoproteins (LPs) in plasma, but data in literature are discordant. We compared different methods for LPs isolation, aiming at finding the best one for studying this association.

Methods: Fresh serum was collected from healthy volunteers. LPs were isolated with different methods, including precipitation with phosphotungstic acid, fast protein liquid chromatography (FPLC), ultracentrifugation using both KBr and iodixanol gradient (IGr). The PCSK9 content of the LP fractions obtained was quantified with ELISA. Cholesterol, APOB and APOA1 were measured using clinical-grade reactives.

Results: In the precipitation-mediated assay, more than 80% of PCSK9 was found in the APOB precipitate. Negligible amount of PCSK9 was detected in the LPs isolated with the KBr ultracentrifugation. PCSK9 was found in the LDL fraction obtained with both IGr ultracentrifugation and FPLC. The percentage of association showed inter and intra individual variability, ranging from 1% to 30% of total recovered PCSK9.

Conclusions: Based on our observations, the PCSK9-LDL association exists and is sensitive to high salt concentrations. IGr ultracentrifugation and FPLC appear to be both suitable for further studies.

 

2.352           Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus

Lemmers, R.E.H., Martens, N.E.M.A., Maas, A.H., van Verk-van Zee, L.C., Leijten, F.P.J., Groot-van Ruijven, C.M., van Hoek, M., Lieverse, A.G., Sijbrands, E.J.G., Haak, H.R., Leenen, P.J.M., Verhoeven, A.J.M., Dik, W.A. and Mulder, M.T.

Atherosclerosis Plus, 44, 43-50 (2021)

 

Background and aims

High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients.

Methods

Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDL_DGUC2), by sequential salt density flotation (HDL_SEQ) or by PEG precipitation (HDL_PEG). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC).

Results

HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 μM, HDL_DGUC2 and HDL_SEQ were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDL_PEG (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDL_DGUC2 for further experiments. With apoA-I at 3.2 μM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC.

Conclusions

The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake.

 

2.353           A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

Perrin-Cocon , L., Vidalain, P-O., Jacquemin, C., Aublin-Gex, A., Olmstead, K., Panthu, B. et al

Communications Biol., 4:4217 (2021)

 

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK⁺/HK2⁻ cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

 

 

2.354           Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma

Jung, J-H., Back, W., Yoon, J., Han, H., Kang, K-W., Choi, B., Jeong, H. et al

Scientific Reports, 11:217 (82021)

 

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

 

2.355           FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Landajuela, A., Braun, M., Rodrigues, C.D.A., Martinez-Cavo, A., Doan, T., Horenkamp, F., Andronicos, A., Shteyn, V., Williams, N.D., Lin, C., Wingreen, N.S., Rudner, D.Z. and Karatekin, E.

PloS Biology, 19(6), e3001314 (2021)

 

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

 

2.356           Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is a Bimodal Process

Garrison, P., Khan, U., Cipriano, M., Bush, P.J., McDonald, J., Sur, A., Myler, P.J., Smith, T.K., Hajduk, S.L. and bangs, J.D.

mBio, 12(4), e01725-21 (2021)

 

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246–257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG⁺ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t_1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t_1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis.

 

2.357           Long-term fasting improves lipoprotein-associated atherogenic risk in humans

Grundler, F., Plonne, D., Mesnage, R., Müller, D., Sirtori, C.R., Ruscica, M., de Toledo, F.W.

Eur. J. Nutr., 60, 4031-4044 (82021)

 

Purpose

Dyslipidemia is a major health concern associated with an increased risk of cardiovascular mortality. Long-term fasting (LF) has been shown to improve plasma lipid profile. We performed an in-depth investigation of lipoprotein composition.

Methods

This observational study included 40 volunteers (50% men, aged 32–65 years), who underwent a medically supervised fast of 14 days (250 kcal/day). Changes in lipid and lipoprotein levels, as well as in lipoprotein subclasses and particles, were measured by ultracentrifugation and nuclear magnetic resonance (NMR) at baseline, and after 7 and 14 fasting days.

Results

The largest changes were found after 14 fasting days. There were significant reductions in triglycerides (TG, − 0.35 ± 0.1 mmol/L), very low-density lipoprotein (VLDL)-TG (− 0.46 ± 0.08 mmol/L), VLDL-cholesterol (VLDL-C, − 0.16 ± 0.03 mmol/L) and low-density lipoprotein (LDL)-C (− 0.72 ± 0.14 mmol/L). Analysis of LDL subclasses showed a significant decrease in LDL1-C (− 0.16 ± 0.05 mmol/L), LDL2-C (− 0.30 ± 0.06 mmol/L) and LDL3-C (− 0.27 ± 0.05 mmol/L). NMR spectroscopy showed a significant reduction in large VLDL particles (− 5.18 ± 1.26 nmol/L), as well as large (− 244.13 ± 39.45 nmol/L) and small LDL particles (− 38.45 ± 44.04 nmol/L). A significant decrease in high-density lipoprotein (HDL)-C (− 0.16 ± 0.04 mmol/L) was observed. By contrast, the concentration in large HDL particles was significantly raised. Apolipoprotein A1 decreased significantly whereas apolipoprotein B, lipoprotein(a), fibrinogen and high-sensitivity C-reactive protein were unchanged.

Conclusion

Our results suggest that LF improves lipoprotein levels and lipoprotein subclasses and ameliorates the lipoprotein-associated atherogenic risk profile, suggesting a reduction in the cardiovascular risk linked to dyslipidemia.

 

2.358           Butyrylcholinesterase–Protein Interactions in Human Serum

Jasiecki, J., Szczoczarz, A., Cysewski, D., Lewandowaki, K., Skowron, P., Waleron, K. and Wasag, B.

Int. J. Mol. Sci., 22(19), 10662 (2021)

 

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein–protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes′ specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

 

2.359           Reconstitution of Functional Integrin αIIbβ3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments

Janke, U., Mitlehner, A., Weide, A., Gutmann, T. and Delcea, M.

Membranes, 11, 499 (2021)

 

The study of the platelet receptor integrin αIIbβ3 in a membrane-mimetic environment without interfering signalling pathways is crucial to understand protein structure and dynamics. Our understanding of this receptor and its sequential activation steps has been tremendously progressing using structural and reconstitution approaches in model membranes, such as liposomes or supported-lipid bilayers. For most αIIbβ3 reconstitution approaches, saturated short-chain lipids have been used, which is not reflecting the native platelet cell membrane composition. We report here on the reconstitution of label-free full-length αIIbβ3 in liposomes containing cholesterol, sphingomyelin, and unsaturated phosphatidylcholine mimicking the plasma membrane that formed supported-lipid bilayers for quartz-crystal microbalance with dissipation (QCM-D) experiments. We demonstrate the relevance of the lipid environment and its resulting physicochemical properties on integrin reconstitution efficiency and its conformational dynamics. We present here an approach to investigate αIIbβ3 in a biomimetic membrane system as a useful platform do dissect disease-relevant integrin mutations and effects on ligand binding in a lipid-specific context, which might be applicable for drug screening.

 

2.360           Lipoprotein Proteomics and Aortic Valve Transcriptomics Identify Biological Pathways Linking Lipoprotein(a) Levels to Aortic Stenosis

Bourgeois, R., Bourgault, J., Despres, A-A., Perrot, N., Guertin, J., Girard, A. et al

Metabolites, 11, 459 (2021)

 

Lipoprotein(a) (Lp(a)) is one of the most important risk factors for the development of calcific aortic valve stenosis (CAVS). However, the mechanisms through which Lp(a) causes CAVS are currently unknown. Our objectives were to characterize the Lp(a) proteome and to identify proteins that may be differentially associated with Lp(a) in patients with versus without CAVS. Our second objective was to identify genes that may be differentially regulated by exposure to high versus low Lp(a) levels in explanted aortic valves from patients with CAVS. We isolated Lp(a) from the blood of 21 patients with CAVS and 22 volunteers and performed untargeted label-free analysis of the Lp(a) proteome. We also investigated the transcriptomic signature of calcified aortic valves from patients who underwent aortic valve replacement with high versus low Lp(a) levels (n = 118). Proteins involved in the protein activation cascade, platelet degranulation, leukocyte migration, and response to wounding may be associated with Lp(a) depending on CAVS status. The transcriptomic analysis identified genes involved in cardiac aging, chondrocyte development, and inflammation as potentially influenced by Lp(a). Our multi-omic analyses identified biological pathways through which Lp(a) may cause CAVS, as well as key molecular events that could be triggered by Lp(a) in CAVS development

 

2.361           The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Zhang, X., Pan, Y-H., Chen, Y., Pan, C., Ma, J., Yan, C., Yu, G. and Ma, J.

1. Biol. Chem., 297(5), 101344 (2021)

 

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

 

 

2.362           Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy

Kojima, W., Yamano, K., Kosako, H., Imai, K., Kikuchi, R., Tanak, k. and Matsuda, N.

Autophagy, 17(8), 2011-2036 (2021)

 

Macroautophagy/autophagy is an intracellular degradation process that delivers cytosolic materials and/or damaged organelles to lysosomes. De novo synthesis of the autophagosome membrane occurs within a phosphatidylinositol-3-phosphate-rich region of the endoplasmic reticulum, and subsequent expansion is critical for cargo encapsulation. This process is complex, especially in mammals, with many regulatory factors. In this study, by utilizing PRKN (parkin RBR E3 ubiquitin protein ligase)-mediated mitochondria autophagy (mitophagy)-inducing conditions in conjunction with chemical crosslinking and mass spectrometry, we identified human BCAS3 (BCAS3 microtubule associated cell migration factor) and C16orf70 (chromosome 16 open reading frame 70) as novel proteins that associate with the autophagosome formation site during both non-selective and selective autophagy. We demonstrate that BCAS3 and C16orf70 form a complex and that their association with the phagophore assembly site requires both proteins. In silico structural modeling, mutational analyses in cells and in vitro phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity.

 

2.363           Self-Assembling Nanoparticle Vaccines Displaying the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Responses in Rhesus Monkeys

Li, H., Guo, L., Zheng, H., Li, J., Zhao, X., Li, J., Liang, Y. et al

Bioconjugate Chem., 32, 1034-1046 (2021)

 

SARS-CoV-2 caused the COVID-19 pandemic that lasted for more than a year. Globally, there is an urgent need to use safe and effective vaccines for immunization to achieve comprehensive protection against SARS-CoV-2 infection. Focusing on developing a rapid vaccine platform with significant immunogenicity as well as broad and high protection efficiency, we designed a SARS-CoV-2 spike protein receptor-binding domain (RBD) displayed on self-assembled ferritin nanoparticles. In a 293i cells eukaryotic expression system, this candidate vaccine was prepared and purified. After rhesus monkeys are immunized with 20 μg of RBD–ferritin nanoparticles three times, the vaccine can elicit specific humoral immunity and T cell immune response, and the neutralizing antibodies can cross-neutralize four SARS-CoV-2 strains from different sources. In the challenge protection test, after nasal infection with 2 × 10⁵ CCID50 SARS-CoV-2 virus, compared with unimmunized control animals, virus replication in the vaccine-immunized rhesus monkeys was significantly inhibited, and respiratory pathology observations also showed only slight pathological damage. These analyses will benefit the immunization program of the RBD–ferritin nanoparticle vaccine in the clinical trial design and the platform construction to present a specific antigen domain in the self-assembling nanoparticle in a short time to harvest stable, safe, and effective vaccine candidates for new SARS-CoV-2 isolates.

 

2.364           The Association of Proprotein Convertase Subtilisin/Kexin Type 9 to Plasma Low-Density Lipoproteins: An Evaluation of Different Methods

Canclini, L., Malvandi, A.M., Uboldi, P., Jabnati, N., Grigore, L., Zambon, A., Baragetti, A. and Catapano, A.L.

Metabolites, 11:861 (2021)

 

Background: Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. Methods: We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Results: Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. Conclusions: The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association.

 

2.365           Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation

Yang, Y., Wu, Z., Wang, l., Zhou, K., Xia, K., Xiong, Q., Liu, L., Zhang, Z., Chapman, E.R., Xiong, Y., Melia, T.J., Karatekin, E., Gu, H. and Lin, C.

Nature Chem., 13, 335-342 (2021)

 

In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA ‘nanobricks’ to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30–130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.

 

2.366           Cryptic splicing events result in unexpected protein products from calpain-10 (CAPN10) cDNA

Ono, Y., Doi, N., Shindo, M., Panico, P. and Salazar, A.M.

BBA-Mol. Cell Res., 1869, 119188 (2022)

 

Calpain-10 (CAPN10) belongs to the calpain superfamily. Genetic polymorphisms of the CAPN10 gene are associated with susceptibility to develop type 2 diabetes mellitus. Although the role of CAPN10 in the pathophysiology of diabetes has been extensively investigated, its biochemical properties are largely unknown. In this report, we made the surprising discovery that CAPN10 cDNA transcripts are subject to cryptic splicing and unexpected protein products were expressed. The same set of splicing products was reproducibly detected in four types of cultured cells including the primary culture of mouse myoblast. At least, one of the products was identical to a natural splicing variant. Sequence analysis of the splicing potential of CAPN10 cDNA, together with mutagenesis studies, resulted in the identification of a powerful splicing acceptor site at the junction of the sequences encoded by exons 9 and 10. We successfully extended the analysis to create expression construct resistant to splicing for both human and mouse CAPN10. The construct allowed us to analyze two major CAPN10 isoforms and reveal their difference in substrate proteolysis and potential cell functions.

These results demonstrate that proteins produced from cDNA do not necessarily reflect the original nucleotide sequence. We provide insight into the property of recombinantly expressed CAPN10 proteins in cultured cells circumventing unexpected protein products.

 

2.367           ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells

Kazuma, Y., Shirakawa, K., Tashiro, Y., Yamazaki, H., Nomura, R. et al

Scientific Reports, 12:2278 (2022)

 

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

2.368           Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy

Lin, J., Yin, L., Xu, X-Z., Sun, H-C., Huang, Z-H., Ni, X-Y., Chen, Y. and Lin, X.

PloS Pathogens, 18(1), e1010204 (2022)

 

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

 

2.369           Ice nucleation in a Gram-positive bacterium isolated from precipitation depends on a polyketide synthase and non-ribosomal peptide synthetase

Failor, K., Liu, H., Llontop, M.E.M., LeBlanc, S., Exkshtain-Levi, N., Sharma, P., Reed, A., Yang, S., Tian, L., lefevre, C.T., Menguy, N., Du, L., Monteil, C.L. and Vinatzer, B.

ISME J., 16, 890-897 (2022)

 

Earth’s radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators, but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens (Lp) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in “pearl chain”-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes.

 

2.370           Hepatic Reduction in Cholesterol 25-Hydroxylase Aggravates Diet-induced Steatosis

Dong, Z., He, F., Yan, X., Xing, Y., Lei, Y., Gao, J., He, M., Li, D., Bai, L., Yuan, Z. and Shyy, J.-Y.

Cell. Mol. Gastroenterol. Hepatol., 13, 1161-1179 (2022)

 

Background & Aims

Cholesterol 25-hydroxylase (Ch25h), converting cholesterol to 25-hydroxycholesterol (25-HC), is critical in modulating cellular lipid metabolism and anti-inflammatory and antiviral activities. However, its role in nonalcoholic fatty liver disease remains unclear.

Methods

Ch25h expression was detected in livers of ob/ob mice and E3 rats fed a high-fat diet (HFD). Gain- or loss-of-function of Ch25h was performed using Ch25h^+/+ (wild type [WT]) mice receiving AAV8-Ch25h or Ch25h knockout (Ch25h^-/-) mice. WT mice fed an HFD were administered with 25-HC. The Ch25h–LXRα–CYP axis was measured in primary hepatocytes isolated from WT and Ch25h^-/- mice.

Results

We found that Ch25h level was decreased in livers of ob/ob mice and E3 rats fed an HFD. Ch25h^-/- mice fed an HFD showed aggravated fatty liver and decreased level of cytochrome P450 7A1 (CYP7A1), in comparison with their WT littermates. RNA-seq analysis revealed that the differentially expressed genes in livers of HFD-fed Ch25h^-/- mice were involved in pathways of positive regulation of lipid metabolic process, steroid metabolic process, cholesterol metabolic process, and bile acid biosynthetic process. As gain-of-function experiments, WT mice receiving AAV8-Ch25h or 25-HC showed alleviated NAFLD, when compared with the control group receiving AAV8-control or vehicle control. Consistently, Ch25h overexpression significantly elevated the levels of primary and secondary bile acids and CYP7A1 but decreased those of small heterodimer partner and FGFR4.

Conclusions

Elevated levels of Ch25h and its enzymatic product 25-HC alleviate HFD-induced hepatic steatosis via regulating enterohepatic circulation of bile acids. The underlying mechanism involves 25-HC activation of CYP7A1 via liver X receptor. These data suggest that targeting Ch25h or 25-HC may have therapeutic advantages against nonalcoholic fatty liver disease.

 

2.371           Recombinant VLPs empower RBM peptides showing no immunogenicity in native SARS-COV-2 protein to elicit a robust neutralizing antibody response

Long, Q., Yang, Y., Yang, M., Bai, H., Sun, W., Yang, X., Huang, W., Li, D. and Ma, Y.

Nanomed.: Nanotechnol. Biol. Med., 41, 102527 (2022)

 

New SARS-COV-2 vaccine strategies are still urgently needed, especially for emerging virus mutations and variants. In this study, we focused on analyzing the antigenicity and vaccine potency of linear peptide epitopes located in receptor binding motif (RBM) of spike (S) protein. Nine 12 to 16-mer overlapping peptides (P1-P9) were synthesized chemically and coupled to carrier protein KLH for the immunization in mice. Four of identified peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. Antisera obtained from VLPs -immunized mice demonstrated strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro. This study indicates that recombinant VLPs empower peptides which display underprivileged antigenicity in native protein to elicit high levels of neutralizing antibody, providing potential epitope candidates and an effective delivery strategy for the development of a multi-epitope vaccine.

 

2.372           Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo

Kass, B., Schemmert, S., Zafiu, C., Kutzsche, j., Bujnicki, T. and Willbold, D.

Cell Report Med., 3, 100630 (2022)

 

The elimination of amyloid beta (Aβ) oligomers is a promising strategy for therapeutic drug development of Alzheimer’s disease (AD). AD mouse models that develop Aβ pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aβ oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aβ oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.

 

2.373           The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain

Loeers, G., Kleene, R., Minguez, M.G. and Schachner, M.

Int. J. Mol. Sci., 23:3554 (2022)

 

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

3. Membranes and Cell Organelles

 

3.1           The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol.

Graham, J., Ford, T. and Rickwood, D. Anal. Biochem., 220, 367-373 (1994)   This paper reports the use of a new density gradient compound, iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient media.  

3.2           A detergent-free method for purifying caveolae membrane from tissue culture cells.

Smart, E.J., Ying, Y-S, Mineo, C. and Anderson, R.G.W. Proc. Natl. Acad. Sci. USA, 92, 10104-10108 (1995)   Current methods for purifying caveolae from tissue culture cells take advantage of the Triton X-100 insolubility of this membrane domain. To circumvent the use of detergents, we have developed a method that depends upon the unique buoyant density of caveolae membrane. The caveolae fractions that we obtain are highly enriched in caveolin. As a consequence we are able to identify caveolae-associated proteins that had previously gone undetected. Moreover, resident caveolae proteins that are soluble in Triton X-100 are retained during the isolation.  

3.3           Acylation targets endothelial nitric‑oxide synthase to plasmalemmal caveolae.

Shaul, P.W. et al

1. Biol. Chem., 271(11), 6518‑6522 (1996)

  Endothelial nitric‑oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent‑free method; eNOS protein was not found in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4‑fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in non‑caveolar plasma membrane. 51‑86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57‑100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS‑7 cells transfected with wild‑type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10‑fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.  

3.4           Clustered folate receptors deliver 5‑methyltetrahydrofolate to cytoplasm of MA104 cells

Smart, E.J., Mineo, C. and Anderson, R.G.W.

1. Cell Biol., 134(5), 1169‑1177 (1996)

  Previously, a high affinity, glycosylphosphatidylinositol‑anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5‑methyltetrahydrofolate in MA104. We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65‑70% of the [³H]folic acid bound to cells in culture. Exposure of cells to the cholesterol‑binding drug, filipin, which is known to uncluster receptors, shifted approximately 50% of the bound [³H]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [³H]folic acid. An mAb directed against the folate receptor also shifted approximately 50% of the caveolae associated [³H]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal  receptor distribution. Concordantly, the mAb inhibited the delivery of  5‑methyl[³H]tetrahydrofolate to the cytoplasm. Receptor bound 5‑methyl [³H]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor‑mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.  

3.5           Localization of epidermal growth factor‑stimulated Ras/Raf‑1 interaction to caveolae membrane.

Mineo, C., James, G.L., Smart, E.J. and Anderson, R.G.W.

1. Biol. Chem., 271(20), 11930‑11935 (1996)

  An essential step in the epidermal growth factor (EGF)‑dependent activation of MAP kinase is the recruitment of Raf‑1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf‑1 is recruited. Caveolae fractions prepared from normal Rat‑1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf‑1 began to appear in caveolae but not in non‑caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf‑1 in this fraction was able to phosphorylate MAP kinase, whereas cytoplasmic Raf‑1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf‑1 to caveolae. Overexpression of Ha‑Ras^v12 caused the recruitment of Raf‑1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA‑5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.  

3.6           A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane.

Smart, E.J., Ying, Ys., Donzell, W.C. and Anderson, R.G.W.

1. Biol. Chem., 271(46), 29427‑29435 (1996)

  Caveolin is a 22‑kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half‑time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non‑caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 mg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting  cholesterol transport is linked to caveolin movement. Caveolae fractions from  cells expressing caveolin were enriched in cholesterol 3‑4‑fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.  

3.7           Subcellular colocalization of the cellular and scrapie prion proteins in caveolae‑like membranous domains.

Vey, M. et al

Proc. Natl. Acad. Sci. USA, 93, 14945‑14949 (1996)   Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrP^Sc) interacts with the substrate cellular PrP (PrP^C) during conversion into nascent PrP^Sc. While PrP^Sc appears to accumulate primarily in lysosomes, caveolae‑like domains (CLDs) have been suggested to be the site where PrP^C is converted into PrP^Sc. We report herein that CLDs isolated from scrapie‑infected neuroblastoma (ScN2a) cells contain PrP^C and PrP^Sc. After lysis of ScN2a cells in ice‑cold Triton X‑100, both PrP isoforms and an N‑terminally truncated form of PrP^C (PrP^C‑II) were found concentrated in detergent‑insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H‑ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N‑hydroxysuccinimide-biotin, both PrP^C and PrP^Sc were found biotinylated in CLD fractions. Similar results on the colocalization of PrP^C and PrP^Sc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrP^C and PrP^Sc are present in CLDs and, thus, support the hypothesis that the PrP^Sc formation occurs within this subcellular compartment.  

3.8           Iodixanol (OptiPrep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes.

Van Veldhoven, P.P., Baumgart, E. and Mannaerts, G.P. Anal. Biochem. 237, 17-23 (1996)                    The suitability of iodixanol, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient. Peroxisomes banded around 30% (w/v) iodixanol (d-1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.  

3.9           Potential chitinase activating factor from yeast cells of Candida albicans.

Jackson, D.J., Saunders, V.A. and Humphreys, A.M. Lett. Appl. Microbiol., 23, 159-162 (1996)   Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30⁰C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a >vacuolar= activating factor in Candida albicans.  

3.10           Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae

Liu, P., Ying, Y., Ko, Y.G  and  Anderson, R.G.W. J Biol. Chem., 271(17), 10299-10303 (1996)   Previously we showed that interleukin lb stimulates the conversion of sphingomyelin to ceramide in the caveolae fraction of normal human fibroblasts.  The ceramide, in turn, blocked platelet-derived growth factor (PDGF) stimulated DNA synthesis.  We now present evidence that the PDGF receptor initiates signal transduction from caveolae.  Cell fractionation and immunocytochemistry show caveolae to be the principal location of PDGF receptors at the cell surface.  Multiple caveolae proteins acquire phosphotyrosine when PDGF binds to its receptor, but the hormone appears to have little effect on the tyrosine phosphorylation of non-caveolae membrane proteins.  Five proteins known to interact with the phosphorylated receptor were found to be highly enriched in caveolae membrane.  PDGF caused the concentration of three of these proteins to significantly increase in the caveolae fraction.  Finally, PDGF stimulated the association of a 190-kDa phosphoprotein with the caveolae marker protein, caveolin.  Therefore, ceramide may modulate PDGF receptor function directly in caveolae.  

3.11           Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA-  and ADP-ribosylation factor (ARF)-regulated isoenzymes.

Provost, J.J. et al.  Biochem. J., 319(2), 285-291 (1996)   Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[g -thio]triphosphate (GTP[S]), with the highest specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.  

3.12           Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells

Majoul, I.V., Bastiaens, P.I.H. and Soling H-D.

1. Cell Biol., 133, 777-789 (1996)

  The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can be used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount  appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 rain after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in the Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3', 5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.  

3.13           Murine SR‑BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N‑glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.

Babitt, J. et al

1. Biol. Chem., 272(20), 13242‑13249 (1997)

  The class B, type I scavenger receptor, SR‑BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor‑mediated endocytosis via clathrin‑coated pits and vesicles. SR‑BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR‑BI (mSR‑BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1‑BS1 cells. mSR‑BI, an approximately 82‑kDa glycoprotein, was initially synthesized with multiple high mannose N‑linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [³H]palmitate and [³H]myristate demonstrated that mSR‑BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol‑ and glycolipid‑rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR‑BI copurified with caveolin‑1, a constituent of  caveolae; and immunofluorescence microscopy demonstrated that mSR‑BI colocalized with caveolin‑1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR‑BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR‑BI‑mediated transfer of lipids between lipoproteins and cells.  

3.14           Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes.

Cartwright, I.J. et al

1. Lipid Res., 38, 531-545 (1997)

  Our aims were (i) to determine which lipids co-localise with newly-synthesised apo-B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilisation and/or translocation of this protein: and (ii) to determine the intracellular sites of assembly of lipids into VLDL. In order to do this, we have developed a new method for the separation of ER derived microsomes on self-generated gradients of iodixanol (OptiPrep). Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles, and the heavier peak contained rough vesicles. Each peak was collected in a number of fractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (SER fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apo-B and VLDL lipids determined by ELISA or high performance thin layer chromatography, respectively. The biosynthetic relationships of apo-B and lipids were investigated, in timed or chase-experiments, by incubation of isolated rabbit hepatocytes with radiolabelled precursors of apo-B or lipids, followed by isolation, and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol and cholesterol ester co-localise with apo-B into the lumen of RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesised triacylglycerol, phospholipid, cholesterol and cholesterol ester were transferred to the lumen of the RER and were chased into the SER lumen. Double-labelling experiments, showed that cholesterol ester produced from newly synthesised cholesterol (labelled with [³H]-mevalonate and [¹⁴C]-oleate) was almost exclusively present in the RER, while cholesterol ester in the SER was labelled only with [¹⁴C]-oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.  

3.15           Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line.

Orlandi, P.A.

1. Biol. Chem., 272(7), 4591-4599 (1997)

  A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A₁ peptide. The latter is an ADP-ribosyltransferase, which activates the a-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p- chloromercuri-benzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% iodixanol gradient, the activity, as measured by CT-A₁ peptide formation localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.  

3.16           The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum.

Yang, M., Ellenberg, J., Bonifacino, J.S. and Weissman, A.M.

1. Biol.Chem., 272(3), 1970-1975 (1997)

  UBC6 is a C-terminal membrane-anchored (type IV) protein, native to Saccharomyces cerevisiae, where it is found in the endoplasmic reticulum. When expressed in mammalian cells, this novel ubiquitin-conjugating enzyme also localizes to the endoplasmic reticulum. UBC6 lacks a lumenal domain and contains no known endoplasmic reticulum retention signals. Analysis of chimeric proteins in which the cytosolic domain of UBC is linked to a heterologous transmembrane domain, or in which the UBC6 transmembrane domain is appended to an unrelated soluble protein, led to the determination that the transmembrane domain of UBC6 plays a dominant role in its compartmental localization. The basis for the transmembrane domain-mediated subcellular targeting of UBC6 was evaluated by lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino acids, which resulted in re-targeting to the Golgi complex. A further increase in length to 26 amino acids allowed this modified protein to traverse the secretory pathway and gain expression at the plasma membrane. These findings are consistent with models in which, in the absence of dominant cytosolic or lumenal targeting determinants, proteins may be sorted within the secretory pathway based on interactions between their transmembrane domains and the surrounding lipid bilayer.  

3.17           Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane

Wu, C., Butz, S., Ying, Y-S and  Anderson, R.G.W.

1. Biol. Chem., 272(6), 3554-3559 (1997)

  Recent evidence suggests that tyrosine kinases are highly organized in caveolae of tissue culture cells.  We now report the isolation of a membrane domain from neuronal plasma membranes that has the biochemical characteristics of caveolae.  A low density membrane (LDM) fraction with the same density as caveolae was highly enriched in tyrosine kinases such as insulin receptors, neurotrophin receptors, Eph family receptors, and Fyn.  Grb2, Ras, heterotrimeric GTP-binding proteins, and Erk2 were also concentrated in the LDM.  Incubation of the LDM fraction at 37°C stimulated the phosphorylation on tyrosine of multiple, resident proteins, whereas the bulk membrane fraction was devoid of tyrosine kinase activity.  The LDM, which makes up ~5-10% of the plasma membrane protein, appears to be organized for signal transduction.  

3.18           Physical association with Ras enhances activation of membrane-bound Raf (RafCAAX)

Mineo, C., Anderson, R.G.W. and White, M.A.

1. Biol. Chem., 272(16), 10345-10348 (1997)

  The transforming activity of artificially membrane targeted Rafl suggests that Ras-mediated recruitment of Rafl to the plasma membrane is an important step in Rafl activation.  Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules.  Growth factor stimulation recruits Rafl to this membrane domain.  Whether Ras simply promotes Rafl association with caveolae membranes or also modulates subsequent activation events is presently unclear.  We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L).  To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V,37G.  Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of rasl2V,37G expression; however, coexpression of rasl2V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase.  These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.  

3.19           Organization of G proteins and adenylyl cyclase at the plasma membrane

Huang, C., Hepler, J.R., Chen, L.T., Gilman, A.G., Anderson, R.G.W. and Mumby, S.M. Mol. Biol. Cell, 8, 2365-2378 (1997)   There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane.  We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane.  These subtractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae.  Immunofluorescence experiments revealed a punctate pattern of G protein a and b subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane.  Partial coincidence of localization of G protein a subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence.  Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically.  Because regulated adenylyl cyclase activity is present in low-density subtractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system.  The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.  

3.20           Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

Liu, P., Ying, Y-S., and  Anderson. R.G.W. Proc. Natl. Acad. Sci., USA, 94, 13666-13670 (1997)   The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete.  Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts.  Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae.  The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.  

3.21           Aminopeptidase I is targeted to the vacuole by a nonclassical vesicular mechanism

Scott, S.V., Baba, M., Ohsumi, Y. and Klionsky, D.J.

1. Cell Biol., 138(1), 37-44 (1997)

  The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments (OptiPrep) demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that the cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.  

3.22           Bisecting GlcNac structures act as negative sorting signals for cell surface Glycoproteins in forskolin-treated rat hepatoma cells

Sultan, A.S. et al

1. Biol. Chem., 272(5), 2866-2872 (1997)

  The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:b-D-mannoside-b-1,4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans.  We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA).  In whole cell lysates, the E-PRA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels.  In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface.  Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and b-glucuronidase, g-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and a-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and g-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.  

3.23           Rapid plasma membrane anchoring of newly synthesized p59^fyn. Selective requirement for NH₂-terminal myristoylation and palmitoylation at cysteine-3.

van’t Hof, W. and Resh, M.D.

1. Cell Biol., 136(5), 1023-1035 (1997)

  Abstract.  The trafficking of Src family proteins after biosynthesis is poorly defined.  Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn.  Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis.  Newly synthesized Src, however, accumulated in the membranes between 20-60 min.  Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1-2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Ga₀-, Ga_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3.  Density gradient analysis colocalized newly synthesized Fyn with plasma membranes.  Interestingly, a 10-20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility.  We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains.  These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.  

3.24           Molecular cloning and expression of a chloride ion channel of cell nuclei

Valenzuela, S.M. et al

1. Biol. Chem., 272(19), 12575-12582 (1997)

  Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles.  However, until recently they were believed not to occur at the nuclear membrane.  In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937.  NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles.  NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.  

3.25           Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled Ga subunits Ga_q and Ga_i in caveolae in DDT₁ MF-2 smooth muscle cells

de Weerd, W.F.C. and Leeb-Lundberg, L.M.F.

1. Biol. Chem., 272(28), 17858-17866 (1997)

  In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT₁MF-2 smooth muscle cells promotes the recruitment and sequestration of the occupied receptors and the receptor-coupled G-protein a subunits Ga_q and Ga_i in caveolae.  Association of ligand receptor complexes and Ga subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae.  Caveolin and Ga subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment.  These complexes were detected with radioligand and by immunoblotting with BK antibodies.  A direct interaction of Ga subunits with caveolin was also indicated by their co-immunoprecipitation.  Immunoelectron microscopy revealed that the enriched caveolin, Ga subunits, and BK receptor complexes were present in structures of 0.1-0.2 mm.  At 4^oC, BK and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment.  Elevation of the temperature to 37°C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling) or 20 min (single-round labeling), and the complexes became acid-resistant.  These conditions also increased the amount of Ga_q and Ga_i. in caveolae with a maximal response at 5-10 min.  In contrast, the NPC17731 receptor complexes remained acid sensitive and dissociated at this temperature, and antagonists did not increase the amount of Ga subunits in caveolae.  These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Ga subunits with caveolae.

3.26           Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers

Molinari, M.et al

1. Biol. Chem., 272(40), 25339-25344 (1997)

  Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker.  Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments.  This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles.  The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.  

3.27           Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component Î-COP

Daro, E., Sheff, D., Gomez, M., Kreis, T. and Mellman, I.

1. Cell Biol., 139(7), 1747-1759 (1997)

  Abstract.  Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells.  We now provide genetic evidence that COPI plays a role in endocytosis in intact cells.  The 1D1F mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit Î-COP.  In addition to exhibiting conditional defects in the secretary pathway, we find that the cells are also defective at mediating endosome-associated functions.  As found for cells microinjected with anti-COPI antibodies, 1D1F cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol.  Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature.  Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited.  In addition, lysosomes redistributed from their typical perinuclear location to the tips of the 1D1F cells.  Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable Î-COP, suggesting that the endocytic defects were not secondary to a block in the secretary pathway.  Importantly, the mutant phenotypes were also corrected by transfection of wild-type Î-COP cDNA demonstrating that they directly or indirectly reflected the Î-COP defect.  Taken together, the results suggest that Î-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

3.28           Identification of caveolin and caveolin-related proteins in the brain

Cameron, P.L., Ruffin, J.W., Bollag, R., Rasmussen, H. and Cameron, R.S.

1. Neurosci., 17(24), 9520-9535 (1997)

  Caveolae are 50-100 nm, nonclathrin-coated, flask-shaped plasma membrane microdomains that have been identified in most mammalian cell types, except lymphocytes and neurons.  To date, multiple functions have been ascribed to caveolae, including the compartmentalization of lipid and protein components that function in transmembrane signaling events, biosynthetic transport functions, endocytosis, potocytosis, and transcytosis.  Caveolin, a 21-24 kDa integral membrane protein, is the principal structural component of caveolae.  We have initiated studies to examine the relationship of detergent-insoluble complexes identified in astrocytes to the caveolin-caveolae compartment detected in cells of peripheral tissues. Immunolocalization studies performed in astrocytes reveal caveolin immunoreactivity in regions that correlate well to the distribution of caveolae and caveolin determined in other cell types, and electron microscopic studies reveal multiple clusters of flask-shaped invaginations aligned along the plasma membrane. Immunoblot analyses demonstrate that detergent insoluble complexes isolated from astrocytes are composed of caveolin-1 a, an identification verified by Northern blot analyses and by the cloning of a CDNA using reverse transcriptase-PCR amplification from total astrocyte RNA.  Using a full-length caveolin-1 probe, Northern blot analyses suggest that the expression of caveolin-1 may be regulated during brain development. Immunoblot analyses of detergent-insoluble complexes isolated from cerebral cortex and cerebellum identify two immunoreactive polypeptides with apparent molecular weight and isoelectric points appropriate for caveolin.  The identification of caveolae microdomains and caveolin-1 in astrocytes and brain, as well as the apparent regulation of caveolin-1 expression during brain development, identifies a cell compartment not detected previously in brain.    

3.29           High-density-lipoprotein subfraction 3 interaction with glycosylphosphatidyl-inositol-anchored proteins

Nion, S. et al Biochem. J., 328, 415-423 (1997)   To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL₃) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C(PI-PLC) significantly decreases specific HDL₃ binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL₃, as revealed by ligand blotting experiments with HDL₃. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL₃-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL₃ inside the vacuolar membrane invaginations. In SK-MES-1 cells, HDL₃ are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL₃ to cells.  

3.30           Reduction of protein disulfide bonds in an oxidizing environment

Majoul, I., Ferrari, D. and Soling, H-D. FEBS Lett., 401, 104-108 (1997)   Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge [Majoul et al (1996) J. Cell Biol. 133, 777-789], although the redox state in the ER favors disulfide formation. We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3. Protein disulfide isomerase (PDI) exerts only a minor accelerating effect. Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction. These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.  

3.31           Calbindin-D_28k in nerve cell nuclei

German, D.C., Ng, M.C., Liang, C.L., McMahon, A and Iacopino, A.M. Neurosci., 81(3), 735-743 (1997)   Calbindin-D_28k is a member of the large EF-hand family of calcium-binding proteins that is believed to function, in part, as a cytosolic calcium buffer. Recent studies have demonstrated that cells containing Calbindin-D_28k are protected from degeneration caused by conditions that elevate intracellular calcium concentration. Since its initial discovery in 1966, Calbindin-D_28k has been localized in the cytoplasm of many neuronal populations, but its nuclear localization has been uncertain. Using light and electron microscopic immunochemistry, and nuclear fractionation methods, we demonstrate localization of Calbindin-D_28k not only in the cytoplasm, but also in the nucleus of rodent midbrain dopaminergic neurons and cerebellar Purkinje cells. The Calbindin-D_28k immunoreactive staining intensity in the nucleus was routinely equal or greater than that in the cytoplasm. Since calcium signals are propagated to the nucleus, where they can regulate gene expression, the existence of nuclear Calbindin-D_28k has important implications for cellular functions.  

3.32           Membrane association of FtsY, the E. coli SRP receptor

DeLeeuw, et al FEBS Lett., 416, 225-229 (1997)   FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SRa), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SRa, FtsY consists of the two major domains: a strongly acidic N-terminal domain (A) and a C-terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associated with the membrane but that the nature of the association differs.

3.33           Role for the target enzyme in deactivation of photoreceptor G protein in vivo

Tsang, S.H., et al. Science, 282(5386), 117-121(1998)   Heterotrimeric guanosine 5’-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3’, 5’-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the g subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.  

3.34           Characterization of a cytosolic heat-shock protein-caveolin chaperone complex.

Uittenbogaard, A., Ying, Y.S. and Smart, E.J.

1. Biol. Chem., 273(11), 6525-6532, (1998)

  Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 mm cyclosporin A or 100 nm rapamycin disrupted the putative transport complex and prevented rapid (10-20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10-20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.  

3.35           Dissection of hepatic receptor-mediated endocytic pathways using self-generated gradients of iodixanol (OptiPrep).

Billington, D., Maltby, P.J. Jackson, A.P. and Graham, J.M. Anal. Biochem., 258, 251-258 (1998).        Iodixanol is a new, non-ionic, iodinated density gradient medium which has the advantage over other similar media in that it rapidly forms self-generated gradients in vertical or near-vertical rotors.  Endocytosis of ^99mTc-labelled neogalactosyl albumin (^99mTc-NGA), a synthetic ligand for the asialoglycoprotein receptor, was studied by administering the ligand as a short pulse to perfused rat livers operating under single pass conditions.  Intracellular processing was arrested at various times after the pulse and the resultant homogenate cleared of nuclei and heavy mitochondria by centrifugation at 3000g for 10 min.  After adjusting to 12.5% (w/v) iodixanol, the 3000g supernatants were centrifuged at 350,000g for 60 min to form the gradients in which early, clathrin-containing vesicles, low-density endosomes and lysosomes were well-resolved.  ^99mTc-NGA bound to the sinusoidal membrane could be partially resolved from clathrin-containing vesicles by inclusion of 1 mM CaCl₂ in the homogenisation and gradient buffers. Two populations of early clathrin-containing vesicles could be resolved by rate-zonal centrifugation in pre-formed iodixanol gradients.  Thus, iodixanol is an excellent density gradient medium for the rapid and efficient resolution of endosome compartments.  

3.36           Subcellular distribution and turnover of presenilins in transfected cells.

Zhang, J. et al

1. Biol. Chem., 273(20), 12436-12442 (1998)

  The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer=s disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilins and their fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon (DE9), we observed only full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the DE9 holoprotein, apparently due to decreased degradation within the ER. Our results suggest that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.  

3.37           Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver.

Wilkinson, J., Higgins, J.A., Fitzsimmons, C. and Bowyer, D.E. Arterioscler. Thromb. Vasc. Biol., 18, 1490-1497 (1998)   Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low density lipoprotein (VLDL), increased low density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by > 70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of d » 1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of d< 1.21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus down regulating the expression of the LDL receptor.  

3.38           Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant non-caveolar membranes.

Waugh, M.G., Lawson, D., Tan, S.K. and Hsuan, J.J.

1. Biol. Chem., 273(27), 17115-17121 (1998)

  This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membranes rafts are the main sites for cellular PtdIns4P production.  

3.39           Rapid enzyme-free preparation of starch-free nuclei from plants facilitates studies of chromatin structure.

Ford, T.C., Baldwin, J.P. and Lambert, S.J. Plant proteins in abiotic stress responses, Plant Protein Club, 1998 Annual Symposium, University of York, p24 (1998)   Several studies of the chromatin structure of normal or stressed plant cells require rapid purification of the cell nuclei under conditions which do not alter the structure of the active or inactive genes under investigation. We have developed a very simple, rapid and enzyme-free method for preparing wheatgerm nuclei by centrifugation on an iodixanol step between densities 1.168 and 1.234 g/ml with a minimal contribution of the iodixanol to solution colligative properties (osmotic pressure). Histone octamers, which are key molecular complexes in the regulation of transcription and which are acetylated in specific lysines in active genes have been purified rapidly from the pure nuclei.    

3.40           Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain.

Smith, R.M., Harada, S., Smith, J.A., Zhang, S. and Jarett, L. Cell. Signalling, 10(5), 355-362 (1998)   The cellular localisation of time- and temperature-dependent ¹²⁵I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2, SOS, Syp, PI 3-kinase, MAP kinase and G_1a were at least 10-fold higher in cell surface-derived, caveolin-enriched fraction than in a cell surface-derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37°C compared with a 3-fold increase in plasma membranes and a 6-fold increase in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDa protein and the 46-kDa Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold-labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin- and IRS-1 containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in the initiating insulin’s signal transduction processes.  

3.41           Purification and characterization of autophagosomes from rat hepatocytes.

Strømhaug, P.E., Berg, T.O., and Seglen, P.O. Biochem. J., 335, 217-224 (1998)   To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-1-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediated-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membraneous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated proteins 1, cation-independent mannose 6-phosphate-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).  

3.42           Presenilin 1 regulates the processing of b-amyloid precursor protein C-terminal fragments and the generation of amyloid b-protein in endoplasmic reticulum and Golgi.

Xia, W. et al Biochemistry, 37(47), 16465-71 (1998)   Progressive cerebral deposition of the amyloid b-protein (Ab) is believed to play a pivotal role in the pathogenesis of Alzheimer’s disease (AD). The highly amyloidogenic 42-residue form of Ab (Ab₄₂) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Ab₄₂ in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Ab. To establish the subcellular localization of the PS1 regulation of APP processing to Ab, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Ab_total and Ab₄₂, Ab₄₂ was the major Ab species in the ER fraction (Ab₄₂:Ab_total ratio 0.5-1.0), whereas absolute levels of both Ab₄₂ and Ab₄₀ were higher in the Golgi fraction and the Ab₄₂:Ab_total ratio was 0.05-0.16 there. Mutant PS1 significantly increased Ab₄₂ levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper g-secretase processing of APP CTFs, and that PS1 mutations augment Ab₄₂ levels principally in Golgi-like vesicles.  

3.43           Role of plasmalemmal caveolae in signal transduction

Shaul, P.W. and Anderson, R.G.W. Am. J. Physiol., 275, 843-851 (1998)   Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules.  They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4°C.  Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules.  This includes C protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules.  More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface.  This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.  

3.44           Targeting of protein Kinase Ca to caveolae

Mineo, C., Ying, Y-S, Chapline, C., Jaken, S. and Anderson, R.G.W.

1. Cell Biol., 141(3), 601-610 (1998)

  Previously, we showed caveolae contain a population of protein kinase Ca (PKCa) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCa and measured the interaction of recombinant PKCa with these membranes. PKCa bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A68-kD PKCa-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCa binding. A 100-aminoacid sequence from the middle of sdr competitively blocked PKCa binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.  

3.45           SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells

Webb, N.R. et al

1. Biol. Chem., 273(24), 15241-15248 (1998)

  The scavenger receptor class B, type I (SR-BI), binds high density lipoprotein (HDL) and mediates selective uptake of cholesteryl ester from HDL and HDL-dependent cholesterol efflux from cells.  We recently identified a new MRNA variant that differs from the previously characterized form in that the encoded C-terminal cytoplasmic domain is almost completely different.  In the present study, we demonstrate that the mRNAs for mouse SR-El and SR-BII (previously termed SR-BI.2) are the alternatively spliced products of a single gene.  The translation products predicted from human, bovine, mouse, hamster, and rat cDNAs exhibit a high degree of sequence similarity within the SR-BII C-terminal domain (62-67% identity when compared with the human sequence), suggesting that this variant is biologically important.  SR-BII protein represents approximately 12% of the total immunodetectable SR-BI/II protein in mouse liver.  Subcellular fractionation of transfected Chinese hamster ovary cells showed that SR-BII, like SR-BI, is enriched in caveolae, indicating that the altered cytoplasmic tail does not affect targeting of the receptor.  SR-BII mediated both selective cellular uptake of cholesteryl ether from HDL as well as HDL-dependent cholesterol efflux from cells, although with approximately 4-fold lower efficiency than SR-BI. In vivo studies using adenoviral vectors showed that SR-BII was relatively less efficient than SR-BI in reducing plasma HDL cholesterol.  These studies show that SR-BII, an HDL receptor isoform containing a distinctly different cytoplasmic tail, mediates selective lipid transfer between HDL and cells, but with a lower efficiency than the previously characterized variant.  

3.46           Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK)

Furuchi, T. and Anderson, R.G.W.

1. Biol. Chem., 273(33), 21009-21104 (1998)

  Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts.  Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen activated protein (MAP) kinase activation.  We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone.  Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src.  Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited.  Despite the reduced amount of c-Raf and Erk2 in the cholesterol depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes.  This was followed by an increase in the amount of active Erk in the cytoplasm.  The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis.  Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis.  These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division.  

3.47           Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells

Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Rodriguez-Boulan,E., Scheller R.H. and Nelson, W.J. Cell, 93, 731-740 (1998)   In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip. We show that Sec6/8 homologs are components of a cytosolic, ~17S complex in nonpolarized MDCK epithelial cells. Upon initiation of calcium-dependent cell-cell adhesion, ~70% of Sec6/8 is rapidly (t_1/2 » 3-6 hr) recruited to sites of cell-cell contact. In streptolyslin-O-permeabilized MDCK cells, Sec6/8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75^NTR to the apical membrane. These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.  

3.48           ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6

Frank, S., Upender, S., Hansen, S.H. and Casanova, J.E.

1. Biol. Chem., 273(1), 23-27(1998)

  ADP-ribosylation factors (ARFS) constitute a family of small monomeric GTPases.  ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis.  Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs).  ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain.  Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi.  However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization.  We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi.  It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of aLb₂ integrins at the plasma membrane of lymphocytes.  

3.49           Retrograde transport of Golgi-localized proteins to the ER

Cole, N.B., Ellenberg, J., Song, J., DiEuliis, D. and Lippincott-Schwarz, J.

1. Cell Biol., 140(1), 1-15 (1998)

  Abstract.  The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment.  Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretary pathway are able to return to the ER folding environment during their lifetime.  Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins.  The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains.  At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules.  Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained.  This occurred over a time period of 15 min-2 h depending on the chimera, and did not require new protein synthesis.  Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex.  This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained.  The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.  

3.50           Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport

Love, H.D., Lin, C.C., Short, C.S. and Ostermann, J.

1. Cell Biol., 140(3), 541-551 (1998)

  Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis -side and exit in transport vesicles budding from the trans-side.  Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretary flow must be recycled constantly by retrograde transport in the opposite direction.  In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells.  We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cistemal membranes.  These vesicles appeared to be depleted of secretary cargo.  They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus.  Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.  

3.51           Caveolin-1 and –2 in the exocytic pathway of MDCK cells

Scheiffele, P. et al

1. Cell Biol., 140(4), 795-806 (1998)

   Abstract.  We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells.  We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER.  The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface.  In the TGN caveolin-l/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-I homooligomers are targeted to the apical side.  Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present.  This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane.  Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein.  Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.  

3.52           Caveolae, plasma membrane microdomains for a-secretase-mediated processing of the amyloid precursor protein

Ikezu, T et al

1. Biol. Chem., 273(17), 10485-10495 (1998)

  Caveolae are plasma membrane invaginations where key signaling elements are concentrated.  In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Ab amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by a-secretase, an as yet unidentified enzyme that cleaves APP within the Ab amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, a-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the a-secretase-mediated proteolysis of APP in vivo.  

3.53           Lipid domain structure of the plasma membrane revealed by patching of membrane components

Harder, T., Scheiffele, P., Verkade, P. and Simons K.

1. Cell Biol., 141(4), 929-942 (1998)

  Abstract.  Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion.  We studied the structure of raft domains in the plasma membrane of non-polarized cells.  Overexpressed plasma membrane markers were evenly distributed in the plasma membrane.  We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers.  For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin.  The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells.  Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains.  In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases.  This view is supported by the finding that cholesterol depletion abrogated segregation.  Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.  

3.54           Cholesterol depletion inhibits the generation of b-amyloid in hippocampal neurons

Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C.G. and Simons, K. Proc. Natl. Acad. Sci. USA, 95, 6460-6464 (1998)   The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease.  During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called b-amyloid (Ab) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APP_sec). It is Ab that accumulates in the brain lesions that are thought to cause the disease.  By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-b-cyclodextrin, we show that the formation of Ab is completely inhibited while the generation of APP_sec is unperturbed.  This inhibition of Ab formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells.  Our results show that cholesterol is required for Ab formation to occur and imply a link between cholesterol, Ab, and Alzheimer's disease.  

3.55           CD14-dependent endotoxin internalization via a macropinocytic pathway

Poussin, C., Foti, M., Carpentier, J.L. and Pugin, J.

1. Biol. Chem., 273(32), 20285-20291 (1998)

  Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein.  This interaction leads to cell activation, but it also promotes LPS internalization and detoxification.  In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway.  In promonocytic THP-1 ells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy 'm sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains.  However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles.  Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae.  With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes.  CD14 was specifically associated with all of these structures.  Radiolabeled LPS internalization paralleled CD14 internalization.  Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.  

3.56           Isolation and characterization of rat liver amphisomes

Berg, T.O., Fengsrud, M., Stromhaug, P.E., Berg, T. and Seglen, P.O.

1. Biol. Chem., 273(34), 21883-21892 (1998)

  Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms.  To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation).  Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the " population.  A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to antophagosomes).  Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion.  The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor).  Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.  

3.57           Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast Saccharomyces cerevisiae

Kametaka, S., Okano, T., Ohsumi, M. and Ohsumi, Y.

1. Biol. Chem., 273(35), 22284-22291 (1998)

  Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apgl4p has a coiled-coil motif at its N terminus region.  We found that overproduction of Apgl4p partially reversed the defect in autophagy induced by the apg6-1 mutation.  The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole.  However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-l mutant, nor did the apgl4 null mutation affect the CPY sorting pathway.  Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vpsl0p, to the late Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J.  Cell Biol 137, 79-92).  Subcellular fractionation indicated that Apgl4p and Apg6p peripherally associated with a membrane structure(s).  Apgl4p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex.  These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway.  Apgl4p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.  

3.58           Annexin XIIIb associates with lipid microdomains to function in apical delivery

Lafont, F., Lecat, S., Verkade, P. and Simons K.

1. Cell Biol., 142(6), 1413-1427 (1998)

  A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells.  Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated.  Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface.  Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts.  We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route.  Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb.  These results suggest that annexin XIIIb directly participates in apical delivery.  

3.59           The Escherichia coli SRP and SecB targeting pathways converge at the translocon

Valent, Q.A. et al The EMBO J., 17(9), 2504-2512 (1998)   Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane.  The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins.  SecB targets the pre-protein to SecA that mediates preprotein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins.  By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane.  The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP.  Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.  

3.60           Functions of lipid rafts in biological membranes

Brown, D.A. and London, E. Annu. Rev. Cell Dev. Biol., 14, 111-136 (1998)   Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties.  Sphingolipid and cholesterol-rich domains may exist as phase-separated “rafts” in the membrane.  We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments.  We also discuss possible functions of lipid rafts in membranes.  Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts.  Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons.  Rafts may also function in sorting and trafficking through the secretary and endocytic pathways.    

3.61           The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells

Kifor, O., Diaz, R., Butters, R., Kifor, I. And Brown, E.M.

1. Biol. Chem., 273(34), 21708-21713 (1998)

  Parathyroid cells have an intracellular machinery for parathyroid hormone (PTH) secretion that is inversely regulated by the extracellular calcium concentration (Ca²⁺_o).  The recently characterized Ca²⁺_o sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor mediating the inhibitory effects of high Ca²⁺_o on PTH secretion.  The CaR's precise cell surface localization and the signal transduction pathway(s) mediating its inhibitory effects on PTH secretion have not been characterized fully.  Here, we demonstrate that the CaR resides within caveolin-rich membrane domains in bovine parathyroid cells.  Chief cells within bovine parathyroid glands exhibit a similar pattern of staining for caveolin-1 and for alkaline phosphatase, a glucosylphosphatidyl-inositol-anchored protein often enriched in caveolae.  Purified caveolin-enriched membrane fractions (CEMF) from bovine parathyroid cells are highly enriched in the CaR and alkaline phosphatase.  Other signaling proteins, including G_q/11, ENOS, and several protein kinase C isoforms (i.e. a, d, and z) are also present in CEMF.  Activation of the CaR by high Ca²⁺_o increases tyrosine phosphorylation of caveolin-1 in CEMF, suggesting that CaR-mediated signal transduction potentially involved in Ca²⁺_o regulated processes in parathyroid cells occur in caveolae-like domains.  

3.62           Palmitoylation of Neurofascin at a Site in the Membrane-Spanning Domain Highly Conserved Among the L1 Family of Cell Adhesion Molecules

Ren, Q. and Bennett, V.

1. Neurochem., 70(5), 1839-1849 (1998)

  This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.  

3.63           The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.

Sheff, D.R., Daro, E.A., Hull, M. and Mellmann, I.

1. Cell Biol., 145(1), 123-139 (1999)

  Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF₄ selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.  

3.64           Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae.

Peterson, T.E. et al. Circulation Res., 85, 29-37 (1999)   Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular home-ostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invagin-ations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifug-ation. Treatment of BAECs with 30 mg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 mmol/L LY83583, a superoxide-generating naptho-quinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine + xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.  

3.65           Purification and characterization of rat hippocamal CA3-dendritic spines associated  with mossy fiber terminals

Kiebler, M.A., Lopez-Garcia, J.C. and Leopold, P.L. FEBS Lett., 445, 80-86 (1999)   We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level.  

3.66           Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol

Plonne, D., Cartwright, I., Linss, W., Dargel, R., Graham, J.M. and Higgins, J.A.Anal. Biochem., 276(1), 88-96 (1999)   A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smooth endoplasmic reticulum (ER), and the rough (ER). Total microsomes were prepared from rat liver by differential centrifugation and resuspended in 20% iodixanol. The microsomal suspension was then layered between a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h. The microsomes distributed in four visible bands. The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyl-transferase (Golgi marker) NADPH-cytochrome c reductase (ER marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Golgi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1.090 g/ml), which contains 68% of the galactosyltransferase activity, consists of vesicles derived from the Golgi. The second broad band in the middle of the tube (density 1.130-1.160 g/ml), which contains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1.220 g/ml) appear to contain two subfractions of vesicles derived from the rough endoplasmic reticulum.  

3.67           Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells

Lafont, F., Verkade, P., Galli, T., Wimmer, C., Louvard, D. and Simons, K. Proc. Natl. Acad. Sci. USA, 96, 3734-3738 (1999)   We have investigated the relationship between apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin-Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.  

3.68           Analysis of CD44-containing lipid rafts: Recruitment of Annexin II and stabilization by the actin cytoskeleton

Oliferenko, S., et al

1. Cell Biol., 146(4), 843-854 (1999)

  CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgS or phalloidin in a semipermeabilized and cholesterol depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.  

3.69           Subcellular distribution of tuberin in cells derived from brain and liver

Yamamoto, Y. and Yeung, R.S. Proc. Amer. Assoc. Cancer Res., 40, #4515 (1999)   Mutations of TSC1 and TSC2 are responsible for the autosomal syndrome of tuberous sclerosis which predisposes carriers to the development of hamartomas and neoplasia. The TSC2 product, tuberin, possesses tumor suppressor activity and in vitro GAP activity towards Rap1 and Rab5. However, the physiological function of tuberin remains poorly understood. Primary sequence analysis suggested a putative transmembrane domain consistent with its abundance in the P100 post nuclear fraction and indirect immunofluorescence demonstrated co-localization with Rap1 and other Golgi markers. In light of the potential role of tuberin in the vesicular trafficking, we studied its subcellular distribution in cells of primary tissues using biochemical fractionation. Microsomal fractions of rat brain and liver were separated on a continuous Iodixanol gradient and analyzed for protein expression of tuberin, rabaptin-5, a TSC2 associated protein, EEA1, and endosomal protein, and TGN38, a trans-Golgi marker. The pattern of expression was similar between tuberin, rabaptin-5 and EEA1 but distinct from that of TGN38. Further, a substantial fraction of tuberin was found free in the cytosol and was re-distributed to the supernatant under conditions of high pH. These findings suggest that tuberin may shuttle between the cytosol and membranous organelles. Further, tuberin localization is not restricted to the Golgi but includes the endosomal compartment. This is consistent with a role of tuberin in vesicular transport.  

3.70           Spatial organization of EGF receptor transmodulation by PDGF

Liu, P. and Anderson, R.G.W. Biochem. Biophys. Res. Commun., 261, 695-700 (1999)   Even though the modulation of EGF receptors by PDGF is well documented, it is not known where on the cell surface cross-talk between the two receptor systems takes place.  The recent finding that both populations of receptors are concentrated in cell surface caveolae suggests that the confinement of the two' receptors to this space might facilitate their interaction.  Here we show that stimulation of PDGF receptors in caveolae with PDGF causes a subpopulation of EGF receptors in the same membrane fraction to become phosphorylated on tyrosine.  Coincident with tyrosine phosphorylation, the binding of EGF to its receptor markedly declines.  Loss of EGF binding is partially blocked by tyrosine kinase inhibitors.  Despite the close proximity of the two receptors in caveolae, we saw no evidence that EGF could stimulate PDGFR tyrosine phosphorylation.  These results suggest that these two receptor systems are highly organized in caveolae.  

3.71           Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesterol ester uptake in THP-1 macrophages

Matveev, S., van der Westhuyzen, D.R. and Smart, E.J.

1. Lipid Res., 40, 1647-1654 (1999)

  Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nM phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation upregulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% ± 1.9% to 38% ± 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.  

3.72           The class B, type I scavenger receptor promotes the selective uptake of high density  lipoprotein cholesterol ethers into caveolae

Graf, G.G., Connell, P.M., van der Westhuyzen, D.R. and Smart, E.J.

1. Biol. Chem., 274(17), 12043-12048 (1999)

  The uptake of cholesterol esters from high density lipoproteins (HDLS) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool.  Cholesterol esters are subsequently internalized to a nonreversible pool.  Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective.  The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin- rich microdomains called caveolae.  Caveolae are directly involved in cholesterol trafficking.  Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE).  Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible  plasma membrane pool of CE.  Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.  

3.73           Polarized distribution of endogenous Racl and RhoA at the cell surface

Michaely, P.A., Mineo, C. Ying, Y-S. and Anderson, R.G.W.

1. Biol. Chem., 274(30), 21430-21436 (1999)

  Racl and RhoA regulate membrane ruffling and stress fiber formation.  Both molecules appear to exert their control from the plasma membrane.  In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface.  We now report that endogenous Racl and RhoA also have a polarized distribution at the cell surface.  Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell.  Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Racl and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA.  We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae.  Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes.  The shift in location was not caused by inactivation of the RhoA effector domain.  Moreover, chimeric proteins containing the C-terminal consensus site for Racl and RhoA prenylation were constitutively targeted to caveolae fractions.  These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.  

3.74           Regulated migration of epidermal growth factor receptor from caveolae

Mineo, C., Gill, G.N. and Anderson, R.G.W.

1. Biol. Chem., 274(43), 30636-30643 (1999)

  In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF.  To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR.  All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae.  By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor.  Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrin coated pits.  Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.  

3.75           Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation

Blair, A., Shaul, P.W., Yuhanna, I.S., Conrad, P.A. and Smart, E.J.

1. Biol Chem., 274(45), 32512-32519 (1999)

  Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide.  Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveolar protein, caveolin.  In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveolar cholesterol in endothelial cells: We found that treatment with 10 mg/ml oxLDL for 60 min causes greater than 90% of eNOS and caveolin to leave caveolae.  Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance.  Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol.  Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae.  Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae.  We conclude that oxLDL-induced depletion of caveolar cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme.  This process may be an important mechanism in the early pathogenesis of atherosclerosis.  

3.76           Subcellular location of enzyme involved in oxidation on n-alkane by Cladosporium resinae

Goswami, P. and Cooney, J.J. Appl. Microbiol. Biotechnol., 51(6), 860-864 (1999)   More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chinitase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 mM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively.  

3.77           Persistent membrane association of activated and depalmitoylated G protein a subunits

Huang, C., Duncan, J.A., Gilman, A.G. and Mumby, S.M. Proc. Natl. Acad. Sci. USA, 96, 412-417 (1999)   Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors.  G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP.  However, the topological fate of activated G protein a subunits is disputed.  One model declares that depalmitoylation of a, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm.  Our data suggest that activation of G protein a subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, a subunits remained bound to the membrane when they were activated with guanosine5'-(3-0-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of a subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.  

3.78           The yeast frataxin homologue mediates mitochondrial iron efflux

Radisky, D.C., Babcock, M.C. and Kaplan, J.

1. Biol. Chem.,274(8), 4497-4499 (1999)

  Mutations in the nuclear gene encoding the mitochondrial protein frataxin are responsible for the neurological disorder Friedreich ataxia (FA).  Yeast strains with a deletion in the frataxin homologue YFH1 accumulate excess iron in mitochondria and demonstrate mitochondrial damage.  We show that in the absence of YFH1, mitochondrial damage is proportional to the concentration and duration of exposure to extracellular iron, establishing mitochondrial iron accumulation as causal to mitochondrial damage.  Reintroduction of YFH1 results in the rapid export of accumulated mitochondrial iron into the cytosol as free, non-heme bound iron, demonstrating that mitochondrial iron in the yeast FA model can be made bioavailable.  These results demonstrate a mitochondrial iron cycle in which Yfhlp regulates mitochondrial iron efflux.  

3.79           Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells

Verkade, P., Harder, T., Lafont, F. And Simons, K.

1. Cell Biol., 148(4), 727-739 (1999)

  In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells.  We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane.  Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T, P. Scheiffele, P. Verkade, and K. Simons. 1998.  J Cell Biol. 929-942), only small (~100 nm) clusters formed on the apical plasma membrane.  Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gpl14 coclustered and were internalized slowly (~10% after 60 min).  Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies.  Upon cholesterol depletion the internalization of PLAP was completely inhibited.  In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.  

3.80           Regulation of b-amyloid secretion by FE65, an amyloid protein precursor-binding  protein

Sabo, S.L. et al

1. Biol. Chem., 274(12), 7952-7957 (1999)

  The principal component of Alzheimer's amyloid plaques, Ab, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP).  FE65 is a brain-enriched protein that binds to APP.  Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear.  We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes.  Moreover, FE65 increases translocation of APP to the cell surface, as well as both aAPP_s and Ab secretion.  The dramatic (4-fold) FE65-dependent increase in Ab secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Ab secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.  

3.81           The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate

Merritt, S.E. et al

1. Biol. Chem., 274(15), 10195-10202 (1999)

  Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH₂-terminal kinase activation.  Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles.  Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system.  Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry.  Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments.  Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7.  That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture.  The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.  

3.82           Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

Meeusen, S. et al

1. Cell Biol., 145(2), 291-304 (1999)

  Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent.  Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid.  MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown.  Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid specific role in maintenance.  Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells.  Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity.  Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgml0lp is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication.  We examined Mgml0lp's role in mtDNA repair.  As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H₂O₂ treatment.  Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.  

3.83           Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophilia raft lipid microdomains

Rietveld, A., Neutz, S., Simons, K. and Eaton, S.

1. Biol. Chem., 274(17), 12049-12054 (1999)

  In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction.  To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes.  Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved.  Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins.  We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction.  Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures.  They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.  

3.84           N-glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in  Madin-Darby canine kidney cells

Benting, J.H., Rietveld, A.G. and Simons, K.

1. Cell Biol., 146(2), 313-320 (1999)

  Glycosyl-phosphatidylinositol (GPI)-anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells.  It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts.  We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells.  The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein.  However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretary proteins, In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly.  Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.  

3.85           Identification and characterization of polycystin-2, the PKD2 gene product

Cai, Y. et al

1. Biol. Chem., 274(40), 28557-28565 (1999)

  PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits.  However, the function of polycystin-2 remains unknown.  We used polyclonal antisera specific for the intracellular NH₂ and COOH termini to identify polycystin-2 as an ~110-kDa integral membrane glycoprotein.  Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins.  Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies.  Polycystin-2 translation products truncated at or after Gly⁸²¹ retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu⁷⁸⁷ additionally traffic to the plasma membrane.  Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells.  The 34-amino acid region GLU⁷⁸⁷-Ser⁸²⁰, containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2.  The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.  

3.86           NK lytic-associated molecule: A novel gene selectively expressed in cells with cytolytic function

Kozlowski, M., Schorey, J., Portis, T., Grigoriev, V. and Kornbluth, J.

1. Immunol., 163, 1775-1785 (1999)

  NK cells are most effective in killing a broad spectrum of primary tumor cells after stimulation with cytokines.  We have cloned a novel gene, designated NKLAM (for NK lytic-associated molecule), whose expression is associated with this cytokine-enhanced process.  NKLAM expression is up-regulated in NK cells by IL-2 and IFNb.  NKLAM is also selectively expressed by activated macrophages and CTL.  Treatment of NK cells and CTL with NKLAM antisense oligonucleotides specifically decreases their cytolytic activity, while having no effect on cell growth.  The NKLAM gene encodes a 62-kDa ring finger-containing protein that localizes to the cytoplasmic granules in NK cells.  Further study of this gene may add to our understanding of cytotoxic processes common to NK cells, CTL, and activated macrophages.  

3.87           Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum

Trombetta, E.S. and Helenius, A. The EMBO J., 18(12), 3282-3292 (1999)   UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle.  We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP.  This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences.  It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate.  By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.  

3.88           Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator

Bradbury, N.A. et al Am. J. Physiol., 276 (Lung Cell. Mol. Physiol., 20), L659-L668 (1999)   Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments.  The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae.  Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column.  Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR.  In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells.  In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae.  These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.  

3.89           Cloning, expression, and cellular localization of a human prenylcysteine lyase

Tschantz, W.R., Zhang, L. and Casey, P.J

1. Biol. Chem., 274(50), 35802-35808 (1999)

  Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus.  These proteins constitute up to 2% of total cellular protein in eukaryotic cells.  The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable.  We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. IL, and Casey, P. J. (1997) J.  Biol Chem. 272,23354-23359).  To facilitate the molecular characterization of this enzyme, its cloning was undertaken.  Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library.  The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs.  Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system.  Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing.  Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.  

3.90           The amyloid precursor protein interacts with G_o heterotrimeric protein within a cell compartment specialized in signal transduction

Brouillett, E. et al

1. Neurosci., 19(5), 1717-1727 (1999)

  The function of the b-amyloid protein precursor (bAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood.  We recently reported the presence of a fraction of bAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction.  To investigate whether bAPP actually interferes with cell signaling, we reexamined the interaction between bAPP and G_o GTPase.  In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22Cl1, an antibody that recognizes an N-terminal bAPP epitope, reduces high-affinity G_o GTPase activity.  This inhibition is specific of G_ao and is reproduced, in the absence of 22Cl1, by the addition of the bAPP C-terminal domain but not by two distinct mutated bAPP C-terminal domains that do not bind G_ao. This inhibition of G_ao GTPase activity by either 22Cl1 or wild-type bAPP cytoplasmic domain suggests that intracellular interactions between bAPP and G_ao could be regulated by extracellular signals.  To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of G_ao and bAPP in CSEM.  We show that inhibition of basal G_ao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of G_ao and bAPP. The regulation of G_ao GTPase activity by bAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of bAPP.  

3.91           ER/Golgi intermediates acquire Golgi enzymes by Bredelfin A – sensitive retrograde transport in vitro

Lin, C-C., Love, H.D., Gushue, J.N., Bergeron, J.J.M. and Osterman, J.

1. Cell. Biol., 147(7), 1457-1472 (1999)

  Secretary proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus.  Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates.  Secretary cargo was arrested at distinct steps of the secretary pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined.  Complementation yield increased after ER exit of secretary cargo and was optimal when transport was blocked at an ER/Golgi intermediate step.  The rapid drop of the complementation yield as secretary cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack.  Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA).  Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step.  Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.        

3.92           Membrane raft microdomains mediate front-rear polarity in migrating cells

Manes, S. et al EMBO J., 18(22), 6211-6220 (1999)   The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-1 (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistri-bution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphat-idylinositol- anchored green fluorescent protein and ephrinBl, to the leading edge. The non-raft proteins  transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF-7 cells, supporting the raft localization requirement for polarization.  IGF-I stimulation  of cholesterol-depleted cells induces projection of multiple pseudopodia over the entire cell  periphery, indicating that raft disruption specifically affects the acquisition of cell polarity but not IGF-I-induced protrusion activity.  Cholesterol depletion inhibits MCF-7 chemotaxis, which is restored by replenishing cholesterol.  Our results indicate that initial segregation between raft and non-raft membrane proteins mediates the necessary, redistribution of specialized molecules for cell migration.  

3.93           Isolated rabbit enterocytes as a model cell system for investigations of chylomicron  assembly and secretion

Cartwright, I.J. and Higgins, J.A.

1. Lipid Res., 40, 1357-1365 (1999)

  A method is described for the isolation of viable enterocytes from rabbit small intestine. The procedure can also be used to isolate populations of epithelial cells from the crypt/villus gradient.  The isolated enterocytes synthesized and secreted apoB-48 and triacylglycerol in particles of the density of chylomicrons.  Secretion was stimulated by addition of bile salt/lipid micelles.  Pulse-chase experiments demonstrated that newly synthesized apoB-48 is degraded intracellularly and that degradation is inhibited by provision of lipid micelles, suggesting that regulation of chylomicron assembly and secretion is broadly similar to that of very low density lipoprotein assembly in hepatocytes.  This procedure for preparation of isolated enterocytes will provide a useful model system for investigation of the molecular details of chylomicron assembly.  

3.94           Caveolin-3 upregulation activates b-secretase-mediated cleavage of the amyloid  precursor protein in Alzheimer’s disease

Nisshiyama, K. et al

1. Neurosci., 19(15), 6538-6548 (1999)

  Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD.  In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro.  Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated b-secretase-mediated processing of APP.  Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes.  Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes.  In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites.  

3.95           Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage

Oh, P. and Schnitzer, J.E.

1. Biol. Chem, 274 (33), 23144-23154 (1999)

  Defining the molecular composition of caveolae is es­sential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-h and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homo­genates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechani­cal disruption. Sonication of plasma membranes, iso­lated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolation to purify caveo­lae. Shearing of silica-coated plasma membranes pro­vides a homogeneous population of caveolae whose con­stituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphos­phatidylinositol-anchored proteins, b-actin, and angio­tensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-la. Caveolin-1b and -2 can also exist in these same caveolae. The isola­tion and analytical procedures as well as the time-de­pendent dissociation of signaling molecules from caveo­lae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent prep­aration for use in the molecular analysis of caveolae.  

3.96           The growth-related, translationally controlled protein P23 has properties of a tubulin protein and associates transiently with microtubules during the cell cycle

Gachet, Y. et al

1. Cell Sci., 112, 1257-1271 (1999)

  The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence. P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In Immunolocalization experiments we find P23 associated with microtubules during G₁, S, G₂ and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with a- and b-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and its alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.  

3.97           VIP36 localisation to the early secretory pathway

Fullekrug, J., Scheiffele, P. and Simons, K.

1. Cell Sci., 112, 2813-2821 (1999)

  VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localized to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al, 1994). Here we provide evidence that endogenous VIP36 is localized to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy, shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalize with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalization with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.  

3.98           Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

Benting, J., Rietveld, A., Ansorge, I. and Simons, K FEBS Lett., 462, 47-50 (1999)   We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei, which contains C14 acyl and alkyl chains, shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.  

3.99           EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains

Bruckner, K. et al Neuron, 22, 511-524 (1999)   Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with “reverse” signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, which we have termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. Our findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands.  

3.100           In vivo imaging of tumors with protease-activated near-infrared fluorescent probes

Weissleder, R., Tung, C-H., Mahmood, U. and Bogdanov, A. Nature Biotech., 17, 375-378 (1999)   We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model In vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous Injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.  

3.101           Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity

Wolfe, M.S., Xia, W., Ostraszewski, B.L., Diehl, T.S., Kimberly, W.T and Selkoe, D.J. Nature 398, 513-517 (1999)   Accumulation of the amyloid-b protein (Ab) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer’s disease. The final step in the generation of Ab from the b-amyloid precursor protein is an apparently intramem­branous proteolysis by the elusive g-secretase(s). The most common cause of familial Alzheimer’s disease is mutation of the genes encoding presenilins 1 and 2, which alters g-secretase activity to increase the production of the highly amyloidogenic Ab₄₂ isoform. Moreover, deletion of presenilin-1 in mice greatly reduces g-secretase activity, indicating that presenilin-1 medi­ates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate resi­dues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Ab production and increases the amounts of the carboxy-terminal fragments of b-amyloid precursor protein that are the substrates of g-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp® Ala mutations also prevented the normal endo­proteolysis of presenilin-1 in the TM6®TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer’s disease and which does not require this cleavage, the Asp 385®Ala mutation still inhibited g-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-I endoproteolysis and g-secretase activity, and suggest that pre­senilin 1 is either a unique diaspartyl cofactor for g-secretase or is itself g-secretase, an autoactivated intramembranous aspartyl protease.  

3.102           Evaluation of the affinity and turnover number of both hepatic mitochondrial and microsomal carnitine acyltransferases: relevance to intracellular partitioning of Acyl-CoAs

Abo-Hashema, K.A., Cake, M.H., Lukas, M.A. and Knudsen, J. Biochemistry, 38, 15840-15847 (1999)   Mitochondrial carnitine palmitoyltransferase I (CPT I) and microsomal carnitine acyltransferase I (CAT I) regulate the entry of fatty acyl moieties into their respective organelles. Thus, CPT I and CAT I occupy prominent positions in the pathways responsible for energy generation in mitochondria and the assembly of VLDL in the endoplasmic reticulum, respectively. Previous attempts to determine the intrinsic kinetic properties of CPT I and CAT I have been hampered by the occurrence of sigmoidal velocity curves. This was overcome, in this study, by the inclusion of recombinant acyl-CoA binding protein in the assay medium. For the first time, we have determined the concentrations of total functional enzyme (E(t)) by specific radiolabeling of the active site, the dissociation constants (K(d)) and the turnover numbers of CPT I and CAT I toward the CoA esters of oleic acid (C18:1) and docosahexaenoic acid (C22:6). The data show that carnitine inhibits CAT I at physiological concentrations which are not inhibitory to CPT I. Thus, carnitine concentration is likely to be a significant factor in determining the partitioning of acyl-CoAs between mitochondria and microsomes, a role which has not been previously recognized. Moreover, the finding that CAT I elicits a lower turnover toward the CoA ester of C22:6 (25 s(-)(1)) than toward that of C18:1 (111 s(-)(1)), while having similar K(d) values, suggests the use of this polyunsaturated fatty acid to inhibit VLDL biosynthesis.  

3.103           The protective role of high-density lipoproteins in atherosclerosis

Mingpeng, S. and Zongli, W. Exp. Gerontol., 34, 539-548 (1999)   Serum high-density lipoprotein level is known to be correlated inversely with the incidence and mortality rates of ischemic heart disease. Although some reports pointed out that in case of hyperalphalipoproteinemia, lesions in the coronary arteries were occasionally found, it is also noticed that in very rare condition, no atheromatous lesions found even in patients with hereditary alphalipoprotein deficiency (Funke et al., 1991). However, clinical surveys have confirmed that high high-density-lipoprotein cholesterol level is favorable in preventing the development of atheroclerotic lesion and high-density lipoprotein together with apolipoprotein AI are currently considered to be the most reliable parameters in predicting the development of atherosclerosis in hyperlipidemia.  

3.104           Membrane structure of caveolae and isolated caveolin-rich vesicles

Westermann, M., Leutbecher, H. and Meyer, H.W. Histochem. Cell Biol., 111, 71-81 (1999)   Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80-120 nm with an open porus of 30-50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30-50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles.            

3.105           Caveolin interacts with Trk A and p75^NTR and regulates neurotrophin signaling pathways

Bilderback, T.R., Gazula, V-R., Lisanti, M.P. and Dobrowsky, R.T.

1. Biol. Chem., 274(1), 257-263 (1999)

  Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75^NTR. We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75^NTR-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75^NTR signaling, and that caveolin can directly interact with p75^NTR. The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75^NTR. In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75^NTR and Trk A in neuronal and glial cell populations.  

3.106           Presence of cholyl- and chenodexoycholyl- coenzyme A thioesterase activity in human liver

Solaas, K., Sletta, R.J., Srreide, O. and Kase, B.F. Scand. J. Clin. Lab. Invest., 60, 91-102 (2000)   In human liver homogenate the formation of bile acid..CoA thioesters is localized both to the microsomal fraction catalysed by an ATP-dependent synthetase and to the peroxisomal fraction catalysed by the thiolase in the last step of the b-oxidative cleavage of the 5b-cholestanoyl side chain. The cytosolic bile acid-CoA:amino acid N-acyltransferase catalyse the conjugation of the CoA-activated bile acids with taurine or glycine prior to secretion into bile. The formation of bile acid-CoA esters is considered the rate-limiting step in bile acid amidation. So far, a bile acid-CoA cleaving activity has not been assessed in the research of bile acid aniidation in human liver. In this work, a bile acid-CoA cleaving activity has been demonstrated at a rate that may influence the concentration of bile acid-CoA thioesters, free bile acids and amidated bile acids within the hepatocyte. Recently, it was shown that free chenodeoxycholic acid, formed by the thioesterase, is the physiological ligand of the farnesoid X receptor.   A multiorganelle distribution of the bile acid-CoA hydrolytic activity was found. In the postnuclear fraction of human liver homogenate, apparent K_m and V_max for the cleavage of choloyl-CoA were 7.7 x 10^-5 mol/L and 3.6 nmol x mg^-1 x min^-1, respectively. The corresponding values for chenodeoxycholoyl-CoA cleavage were 7.1 x 10^-5 mol/L and 4.8 nmol x mg^-1 x min^-1. Hydrolytic activities were detected in the microsomal and the peroxisomal fractions where the bile acid-­CoA esters are formed as well as in cytosol housing the N-acyltransferase activity. Compared to the bile acid-CoA synthetase activities, the hydrolytic activities were considerably higher, both in the postnuclear fraction and in the microsomal fraction. The thioesterase activities were in the same range as detected for the N-­acyltransferase activities both in the postnuclear fraction and in the cytosolic fraction. The mere presence of thioesterase in microsomes, peroxisomes and cytosol seems counterproductive to bile acid amidation. The thioesterases may have an indirect regulatory function on the bile acid synthesis and are important for the regulation of bile acid synthesis by providing free chenodeoxycholic acid, the most potent activator of the farnesoid X receptor.  

3.107           Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes

Hashiramoto, M. and James, D.E. Mol. Cell Biol., 20(1), 416-427 (2000)   Insulin regulates glucose transport in muscle and adipose tissue by triggering translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.  

3.108           Activated cardiac adenosine A₁ receptors translocate out of caveolae

Lasley, R.D., Narayan, P., Uittenbogaard, A. and Smart, E.J.

1. Biol. Chem., 275(6), 4417-4421 (2000)

  The cardiac affects of the purine nucleoside, adenosine, are well known.  Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects.  These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane.  Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation.  In this study, we tested the hypothesis that adenosine A₁ receptors would translocate to caveolae in the presence of agonists.  Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67±5% of adenosine A₁ receptors were isolated with caveolae.  However, incubation with the adenosine A₁ receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A₁ receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3dipropylxanthine.  An adenosine A₁ receptor agonist did not alter the localization of A₁ receptors to caveolae. These data suggest that the translocation of A₁ receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A₁ receptors are associated with few direct effects.  

3.109           High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae

Uittenbogaard, A., Shaul, P.W., Yuhanna, I.S., Blair, A. and Smart, E.J.

1. Biol. Chem., 275(15), 11278-11283 (2000)

  Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation.  In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function.  We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immuno-precipitate with caveolin-1.  Co-incubation of cells with 10 ug/ml high density lipoprotein (HDL) prevented oxLDL induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 ug/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL induced  redistribution of eNOS.  SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL induced depletion of caveola cholesterol We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation.  Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.  

3.110           Gastropod mollusc aliphatic alcohol oxidase: subcellular localisation and properties

Grewal, N., Parveen, Z., Large, A., Perry, C. and Connock, M. Comp. Biochem. Biophys., 125, 543-554 (2000)   The digestive gland and other tissues of several species of terrestrial gastropod mollusc contain an aliphatic alcohol oxidase activity (EC1.1.3.13). The enzyme is FAD dependent, consumes oxygen and generates hydrogen peroxide and the corresponding aldehyde.  Saturated primary alcohols arc favoured as substrates with octanol preferred with an apparent K_m of 3-4 mM.  The activity is clearly distinguishable from previously reported molluscan aromatic alcohol oxidase (EC1.1.3.7) on the basis of FAD dependence, sensitivity to heat treatment and hi-h salt concentration and with regard to substrate preferences.  The aliphatic alcohol oxidase is membrane associated and most likely localised to the endoplasmic reticulum.  Extraction of membranes with 1% Igipal solubilises the enzyme in active form.  This enzyme is a further example of an oxidase apparently restricted to molluscs.  

3.111           CCC1 suppresses mitochondrial damage in the yeast model of Friedreich’s ataxia by limiting mitochondrial iron accumulation

Chen, O.S. and Kaplan J.

1. Biol. Chem., 275(11), 7626-7632 (2000)

  Deletion of YFH1 in Saccharomyces cerevisiae leads to a loss of respiratory competence due to excessive mitochondrial iron accumulation. A suppressor screen identified a gene, CCC1, that maintained respiratory function in a Dyfh1 yeast strain regardless of extracellular iron concentration. CCC1 expression prevented excessive mitochondrial iron accumulation by limiting mitochondrial iron uptake rather than by increasing mitochondrial iron egress. Expression of CCC1 did not result in sequestration of iron in membranous compartments or cellular iron export. CCC1 expression in wild type cells resulted in increased expression of the high affinity iron transport system composed of FET3 and FTR1, suggesting that intracellular irons is not sensed by the iron-dependent transcription factor Aft1p. Introduction of AFT1^up, a constitutive allele of the iron transcription factor, AFT1, that also leads to increased high affinity iron transport did not prevent Dyfh1 cells from becoming respiratory-incompetent. Although the mechanism by which CCC1 expression affects cytosolic iron is not known, the data suggest that excessive mitochondrial iron accumulation only occurs when cytosolic free iron levels are high.  

3.112           Subcellular localization of presenilins: association with a unique membrane pool in cultured cells

Kim, S.H., Lah, J.J., Thinakaran, G., Levey, A. and Sisodia S.S Neurobiol. Dis., 7(2), 99-117 (2000)   We have investigated the subcellular distribution of presinilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol –based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast PS1 and PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.  

3.113           Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes

Sims, A.C., Ostermann, J. and Denison, M.R.

1. Virol., 74(12), 5647-5654 (2000)

  The coronavirus replicase gene (gene1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopy studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of the populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.  

3.114           Characterization of isolated acidocalcisomes of Trypanosoma cruzi

Scott, D.A. and Docampo, R.

1. Biol. Chem., 275(31), 24215-24221 (2000)

  The acidocalcisome is an acidic calcium store in trypanosomatids, with a vacuolar-type proton-translocating pyrophosphatase (V-H⁺-PPase) located in the membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisomes from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000xg) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca²⁺ uptake (Ca²⁺-ATPase) activity was detectable in isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified and rate of Ca²⁺ uptake increased. Use of the indicator Oxonol VI showed that PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H⁺-ATPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H⁺-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H⁺-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H⁺-ATPase and V-H⁺-PPase activities are present in the same Ca²⁺-containing compartment.  

3.115           The existence of a lysosomal redox chain and the role of ubiquinone

Gille, L. and Nohl, H. Arch. Biochem. Biophys., 375(2), 347-354 (2000)   Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes.  Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles.  The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes.  In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation.  The purity of our preparation was verified by estimation of the respective marker enzymes.  Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques.  UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes.  Furthermore, a b -type cytochrome and a flavin-adenine dinucleotide (FAD) were identified as other potential electron carriers.  Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes.  Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O₂ acts as a terminal acceptor of this redox chain.  DMPO/OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen.  The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen.  By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes.  In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.  

3.116           Caveolar structure and protein sorting are maintained in NIH cells independent of glycosphingolipid depletion

Shu, L et al Arch. Biochem. Biophys., 373(1), 83-90 (2000)   Glycosphingolipids have been proposed to be critical components of cluster lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20% and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.  

3.117           Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast

Bagnat, M., Keranen, S., Shevchenko, A., Shevchenko, A. and Simons, K. Proc. Natl. Acad. Sci.,. USA, 97(7), 3254-3259 (2000)   Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec 18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.  

3.118           Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Heino, S. et al Proc. Natl. Acad. Sci., USA, 97(15), 8375-8380 (2000)   In this study, we compared the transport of newly synthesized cholesterol with that of influenza hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.  

3.119           Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae

Uittenbogaard, A. and Smart, E.J.

1. Biol. Chem., 275(33), 25595-25599 (2000)

  We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10-20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.  

3.120           Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum

Pedrazzini, E., Villa, A., Longhi, R., Bulbarelli, A. and Borgese, N.

1. Cell Biol., 148(5), 899-913 (2000)

  Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two.  Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretary pathway by largely unknown mechanisms.  Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins.  We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms.  Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation.  These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.  

3.121           The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles

Elkind., N.B., Walch-Solimena, C. and Novick, P.J.

1. Cell Biol., 149(1), 95-110 (2000)

  Sec2p is required for the polarized transport of secretory vesicles in S cerevisiae. The Sec2p NH₂ terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips.  Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown.  We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate.  Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips.  A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization.  Sec2p localization depends on actin, Myo2p and Seclp, Sec6p, and Sec9p function.  Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes.  Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37°C but unaffected at 25°C.  Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association.  In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.  

3.122           Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake

Yun, C-W. et al

1. Biol. Chem., 275(14), 10709-10715 (2000)

  In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aftl. cDNA microarrays were used to identify new iron and AFT1-regulated genes.  Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters.  These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease.  Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine bound iron and growth on ferrioxamine as an iron source.  The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments.  Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pepl2.  In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases.  These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.  

3.123           Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft  domains

Lockwich, T.P. et al

1. Biol. Chem., 275(16), 11934-11942 (2000)

  Trpl has been proposed as a component of the store-operated Ca²⁺ entry (SOC) channel.  However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood.  We have examined possible molecular interactions involved in the regulation of SOC and Trpl and report here for the first time that Trpl is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains.  Endogenous hTrpl and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes.  Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trpl and inhibited carbachol-stimulated Ca²⁺ signaling.  Importantly, thapsigargin stimulated Ca²⁺ influx, but not internal Ca²⁺ release, and inositol 1,4,5-triphosphate (IP₃) stimulated I_soc were also attenuated.  Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrpl, caveolin-1, Ga_q/11 and IP₃ receptor-type 3 (IP₃R3).  These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca²⁺ signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC.  Importantly, we have shown that Trpl is also localized in this microdomain where it interacts with one or more components of this complex, including IP₃ R3.  This finding is potentially important in elucidating the physiological function of Trp.  

3.124           Enterotoxigenic Escherichia coli secretes active heat-labile enteroxin via outer membrane vesicles

Horstman, A.L. and Kuehn, M.J.

1. Biol. Chem., 275(17), 12489-12496 (2000)

  Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth.  Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells.  Enterotoxigenic E.coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation.  Vesicle protein profiles were similar but not identical to outer membranes and differed between strains.  Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal.  Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles.  Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles.  In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains.  

3.125           Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR–mediated retinal and macular degeneration

Mata, N.L., Weng, J. and Travis, G.H. Proc. Natl. Acad. Sci. USA, 97(13), 7154-7159 (2000)   Increased accumulation of lipofuscin in cells of the retinal pigment epithelium (RPE) is seen in several forms of macular degeneration, a common cause of blindness in humans. A major fluorophore of lipofuscin is the toxic bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E).  Previously, we generated mice with a knockout mutation in the abcr gene.  This gene encodes rim protein (RmP), an ATP-binding cassette transporter in rod outer segments.  Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over controls.  Here, we identify three precursors of A2E in ocular tissues from abcr/mice and humans with ABCR-mediated recessive macular degenerations.  Our results corroborate the scheme proposed by C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon & J. Sparrow [Proc. Natl. Acad. Sci. USA (1998) 95,14609-14613], for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-trans-RAL) with phosphatidylethanolamine to form a Schiff base; (ii) condensation of the amine product with a second all-trans-RAL to form a bis-retinoid; (iii) oxidation to yield a pyridinium salt; and (iv) hydrolysis of the phosphate ester to yield A2E.  The latter two reactions probably occur within RPE phagolysosomes.  As predicted by this model, formation of A2E was completely inhibited when abcr / mice were raised in total darkness.  Also, once formed, A2E was not eliminated by the RPE.  These data suggest that humans with retinal or macular degeneration caused by loss of RmP function may slow progression of their disease by limiting exposure to light. The precursors of A2E identified in this study may represent pharmacological targets for the treatment of ABCR-mediated macular degeneration.  

3.126           Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Plant PJ. et al

1. Cell Biol., 149(7), 1473-1483 (2000).

  Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain.  We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells.  To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface.  Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ~35-40-kD protein that we identified as annexin XIII using mass spectrometry.  Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment.  In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 mM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane.  Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+ -dependent manner, as determined by detergent extraction and floatation assays.  These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.  

3.127           Mutation of conserved aspartates affects maturation of both aspartate mutant and  endogenous presenilin 1 and presenilin 2 complexes

Yu, G. et al

1. Biol. Chem., 275(35), 27348-27353 (2000)

  Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW complexes.  During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments.  Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of b-amyloid precursor protein and Notch.  We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes.  Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes.  These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes.  

3.128           ³¹P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major

Moreno, B. et al

1. Biol. Chem., 275(37), 28356-28362 (2000)

  High resolution ³¹P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a ¹H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, the causative agents of African sleeping sickness, Chagas’disease, and leishmaniasis.  Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds.  The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. ³¹P NMR spectra of purified T. brucei, T. cruzi and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates.  In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.  

3.129           Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation

Pol, A., Lu, A., Pons, M., Peiro, S. and Enrich, C.

1. Biol. Chem., 275(39), 30566-30572 (2000)

 

The endocytic compartment of eukaryotic cells is a complex intracellular structure involved in sorting, processing, and degradation of a great variety of internalized molecules.  Recently, the uptake through caveolae has emerged as an alternative internalization pathway, which seems to be directly related with some signal transduction pathways.  However, the mechanisms, molecules, and structures regulating the transport of caveolin from the cell surface into the endocytic compartment are largely unknown.  In this study, normal quiescent fibroblasts (normal rat kidney (NRK)) were used to demonstrate that epidermal growth factor causes partial redistribution of caveolin from the cell surface into a cellubrevin early endocytic compartment.  Treatment of NRK cells with cytochalasin D or latrunculin A inhibit this pathway and the concomitant activation of Mek and mitotic-activated protein (MAP) kinase; however, if cells were pre-treated with filipin, cytochalasin D does not inhibit the phosphorylation of MAP kinase induced by epidermal growth factor.  From these results we conclude that in NRK cells the intact actin cytoskeleton is necessary for the EGF-mediated transport of caveolin from the cell surface into the early endocytic compartment and the activation of MAP kinase pathway.  

3.130           Dystrophin associates with a caveolae of rat cardiac myocytes

Doyle, D.D. et al Circulation Res., 87, 480-488 (2000)

 

The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan.  Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan.  Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies.  In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan.  Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies.  To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes.  Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped.  Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae.  These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex.  

3.131           Chemical stimulation of synaptosomes modulates a-Ca²⁺/calmodulin-dependent protein kinase II mRNA association to polysomes

Bagni, C., Mannucci, L., Dotti, C.G. and Amaldi, F.

1. Neurosci., 20 RC76, 1-6 (2000)

 

The presence of specific mRNAs in dendrites and at synapses is well established, but a direct and reliable demonstration that they are associated with polysomes is still missing.  To address this point we analyzed the polysomal association of the mRNAs for the a-subunit of Ca²⁺ /calmodulin-dependent protein kinase II (a-CaMKII), for type 1 inositol 1,4,5-trisphosphate receptor (InsP3Rl) and for the activity-regulated cytoskeleton-associated protein (Arc) in a synaptosomal preparation devoid of contaminating material from neuronal and glial perikarya.  We show that a fraction of a-CaMKII, InsP3Rl, and Arc mRNAs present in synaptosomes is indeed associated with polysomes. Moreover, we show that polysomal association of a-CaMKII mRNA, but not InsP3Rl and Arc mRNAs, increases with depolarization of the synaptosomal membrane.  Finally, we show that the synthesis of a-CaMKII protein increases with stimulation.  Dendritic mRNA recruitment onto polysomes in response to synaptic stimulation might represent one of the mechanisms underlying the processes of learning and memory.  

3.132           Mutant presenilin 1 increases the levels of Alzheimer amyloid b-peptide Ab42 in late compartments of the constitutive secretory pathway

Petanceska, S., Seeger, M., Checler, F. and Gandy, S.

1. Neurochem., 74, 1878-1884 (2000)

  Mutations in the presenilin 1 (PS1) gene are associated with autosomal dominant, early-onset, familial Alzheimer's disease and result in increased release of the hyperaggregatable 42-amino acid form of the amyloid b-peptide (Ab42).  To determine which subcellular compartments are potential source(s) of released Ab42, we compared the levels and spatial segregation of intracellular Ab40 and Ab42 peptides between N2a neuroblastoma cells doubly transfected with the "Swedish" familial Alzheimer's disease-linked amyloid precursor protein variant and either wild-type PS1 (PS1^wt) or familial Alzheimer's disease-linked D9 mutant PS1 (PS1^D9).  As expected, PSl^D9-expressing cells had dramatically higher levels of intracellular Ab42 than did cells expressing PS1^wt.  However, the highest levels of Ab42 colocalized not with endoplasmic reticulum or Golgi markers but with rab8, a marker for trans-Golgi network (TGN)-to-plasma membrane (PM) transport vesicles.  We show that PS1 mutants are capable of causing accumulation of Ab42 in late compartments of the secretary pathway, generating there a readily releasable source of Ab42.  Our findings indicate that PS1 "bioactivity" localizes to the vicinity of the TGN and/or PM and reconcile the apparent discrepancy between the preponderant concentration of PS1 protein in proximal compartments of the secretary pathway and the recent findings that PS1 "bioactivity" can control g-secretase-like processing of another transmembrane substrate, Notch, at or near the PM.  

3.133           Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland

Nemeth, B.A., Tsang, S.W.Y., Geske, R.S. and Haney, P. Pediatr Res., 47, 444-450 (2000)   Lactose, the major carbohydrate of human milk, is synthesized in the Golgi from glucose and UDP-galactose.  The lactating mammary gland is unique in its requirement for the transport of glucose into Golgi.  Glucose transporter-1 (GLUT1) is the only isoform of the glucose transporter family expressed in mammary gland.  In most cells, GLUT1 is localized to the plasma membrane and is responsible for basal glucose uptake; in no other cell type is GLUT1 a Golgi resident.  To test the hypothesis that GLUT1 is targeted to Golgi during lactation, the amount and subcellular distribution of GLUT1 were examined in mouse mammary gland at different developmental stages.  Methods including immunohistochemistry, immunofluorescence, subcellular fractionation, density gradient centrifugation, and Western blotting yielded consistent results.  In virgins, GLUT1 expression was limited to plasma membrane of epithelial cells.  In late pregnant mice, GLUT1 expression was increased with targeting primarily to basolateral plasma membrane but also with some intracellular signal. During lactation, GLUT1 expression was further increased, and targeting to Golgi, demonstrated by colocalization with the 110-kD coatomer-associated protein b-COP, predominated.  Removal of pups 18 d after delivery resulted in retargeting of GLUT1 from Golgi to plasma membrane and a decline in total cellular GLUT1 within 3 h. In mice undergoing natural weaning, GLUT1 expression declined.  Changes in the amount and targeting of GLUT1 during mammary gland development are consistent with a key role for GLUT1 in supplying substrate for lactose synthesis and milk production.  

3.134           Membrane raft microdomains mediate lateral assemblies for HIV-1 infection

Manes, S. et al EMBO reports, 1(2), 190-196 (2000)   HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gpl2O-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors.  Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment.  This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.  

3.135           Subcellular organization of bile acid amidation in human liver: a key in regulating the biosynthesis of bile salts

Solaas, K., Ulvestad, A., Soreide, O. and Kase, B.F.

1. Lipid Res., 41, 1154-1162 (2000)

 

To extend our knowledge of how the synthesis of free bile acids and bile salts is related within the hepatocytes, bile acid-CoA:amino acid N-acyltransferase and bile acid-CoA thioesterase activities were measured in subcellular fractions of human liver homogenates.  Some bile acids, both conjugated and unconjugated, have been reported to be natural ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor.  The conversion of [¹⁴C]choloyl-CoA and [¹⁴C]chenodeoxycholoyl-CoA into the corresponding tauro- and glyco-bile acids or the free bile acids was measured after high-pressure liquid radiochromatography.  There was an enrichment of the N-acyltransferase in the cytosolic and the peroxisomal fraction.  Bile acid-CoA thioesterase activities were enriched in the cytosolic, peroxisomal, and mitochondrial fractions.  The highest amidation activities of both choloyl-CoA and chenodeoxycholoyl-CoA were found in the peroxisomal fraction (15-58 nmol/mg protein/min).  The K_m, was higher for glycine than taurine both in cytosol and the peroxisomal fractions These results show that the peroxisomal de novo synthesis of bile acids is rate limiting for peroxisomal amidation, and the microsomal bile acid-CoA synthetase is rate limiting for the cytosolic amidation.  The peroxisomal location may explain the predominance of glyco-bile acids in human bile.  Both a cytosolic and a peroxisomal bile acid-CoA thioesterase may, influence the intracellular levels of free and conjugated bile acids.  

3.136           Human bleomycin hydrolase regulates the secretion of amyloid precursor protein

Lefterov, I.M., Koldamova, R.P. and Lazo, J.S. FASEB J., 14, 1837-1847 (2000)   Human bleomycin hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease.  We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, b amyloid (Ab).  We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization.  Metabolic labeling and pulse-chase experiments, showed that ectopic hBH expression increased secretion of soluble APPa/b products without changing the half-life of cellular APP. We also observed that this increased Ab secretion was independent of hBH isoforms.  Our findings suggest a regulatory role for hBH in APP processing pathways.    

3.137           Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Antonenkov, V.D., Croes, K., Waelkens, E., Van Veldhoven, P.P. and Mannaerts, G.P. Eur. J. Biochem., 267, 2981-2990 (2000)   Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

 

3.138           Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane

Gimpl, G. and Fahrenholz, F. Eur. J. Biochem., 267, 2483-2497 (2000)   We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane.  For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells, To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor.  Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains.  In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched.  In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t, » threefold) stability at 37°C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes.  Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function.  The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso.  In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.  

3.139           Involvement of gangliosides in GPI-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid rafts

Kasahara, K. et al

1. Biol. Chem., 275(44), 34701-34709 (2000)

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol (GPI)-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the src-family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was GPI-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated crosslinking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for src-family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1 mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1 mediated signaling in lipid rafts.  

3.140           Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates

Liu, P., Wang, P-y., Michaely, P., Zhu, M. and Anderson, R.G.W.

1. Biol. Chem., 275(41), 31648-31654 (2000)

  Platelet-derived growth factor receptor b (PDGFRb) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown.  We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners.  The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.  

3.141           Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation and autophagy

Kegel, K.B. et al

1. Neurosci., 20(19), 7268-7278 (2000)

  An expansion of polyglutamines in the N terminus of huntingtin causes Huntington’s disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons.  How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur.  We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink.  Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin.  Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes mitochondria, and nuclear membranes.  Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes.  Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles.  We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.  

3.142           The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to  Alzheimer’s disease-associated neurodegeneration and oxidative stress

Greeve, I. et al

1. Neurosci., 20(19), 7345-7352 (2000)

  In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration.  To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display.  We identified seladin-1, a novel gene, which was down regulated in large pyramidal neurons in vulnerable regions in AD but not control brains.  Seladin- 1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans.  Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases.  In human control brain, seladin-1 was highly expressed in almost all neurons.  In PC12 cell clones that were selected for resistance against AD-associated amyloid-b peptide (Ab)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells.  Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Ab-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death.  In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner.  These results establish that seladin-1 is an important factor for the protection of cells against Ab toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death, Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.  

3.143           Carboxyl-terminal fragments of Alzheimer b-amyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1 deficient cells

Chen, F. et al

1. Biol. Chem., 275, 36794-36802 (2000)

  Absence of functional presenilin 1 (PS1) protein leads to loss of g-secretase cleavage of the amyloid precursor protein (bAPP), resulting in a dramatic reduction in amyloid b peptide (Ab) production and accumulation of a- or b-secretase-cleaved C-terminal fragments of bAPP (a- or b-CTFs). The major C-terminal fragment (CTF) in brain was identified as bAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Ab(11-40). In PS1^-/- murine neurons and fibroblasts expressing the loss-of-function PS1_D385A mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1^-/- neurons as compared to PS1_D385A mutant fibroblasts. However, there was no obvious redistribution of full-length bAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1^-/- neurons (as in normal cells) trafficking of bAPP to the Golgi compartment is necessary before a-and b-secretase cleavage occurs. Thus, while we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual g-secretase catalytic activity or in other functions indirectly related to g-secretase catalysis (e.g. an activator of g-secretase, a substrate-adaptor for g-secretase, or delivery of g-secretase to bAPP-containing compartments.  

3.144           Differential targeting of b-adrenergic receptor subtypes and adenyl cyclase to cardiomyocyte caveolae: A mechanism to functionally regulate the cAMP signaling  pathway

Rybin, V.O., Xu, X., Lisanti, M.P. and Steinberg, S.F.

1. Biol. Chem., 275(52), 41447-41457(2000)

  Differential modes for b₁ and b₂-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdo­mains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in car­diomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in b₂-ARs, Ga_i, protein kinase A, RIIa subunits, caveolin-3, and flotillins (caveolin functional homologues); b₁-ARs, m₂-muscarinic cholinergic receptors, Ga_s and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIa subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface b₂-ARs lo­calize to caveolae in cardiomyocytes and cardiac fibro­blasts (with markedly different b₂-AR expression levels), indicating that the fidelity of b₂-AR targeting to caveo­lae is maintained over a physiologic range of b₂-AR ex­pression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of b₂-ARs (but not b₁-ARs) in caveolae. Other studies show co-immunopre­cipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. How­ever, caveolin is not required for adenylyl cyclase tar­geting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Neverthe­less, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accu­mulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the b-AR signaling cascade represent impor­tant modifiers of cAMP-dependent signaling in the heart.    

3.145           Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling  assemblies

Harder, T. and Kuhn, M.

1. Cell Biol., 151(2), 199-207 (2000)

  Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes.  Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction.  Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells.  We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins.  We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.  In contrast, other raft-associated molecules, including protein tyrosine kinases Lek and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.  Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/ TCR assemblies form a structural scaffold for TCR signal transduction proteins.  Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.  

3.146           Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid b-protein generation

Xia, W. et al Proc. Natl. Acad. Sci., 97(16), 9299-9304 (2000)   An unusual intramembranous cleavage of the b-amyloid precursor protein (APP) by g-secretase is the final step in the generation of amyloid b-peptide (Ab).  Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Ab production by g-secretase.  Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of g-secretase, can be coimmunoprecipitated with both PS1 and PS2.  PS/C83 complexes were detected in cells expressing endogenous levels of PS.  The complexes accumulate when g-secretase is inactivated either pharmacologically or by mutating the PS aspartates.  PS1/ C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions.  In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of g-secretase, occurred earlier in endoplasmic reticulum-rich vesicles.  The major portion of intracellular Ab at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Ab levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine.  Furthermore, de novo generation of Ab in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate.  Thus, PSs are complexed with the g-secretase substrates C83 and C99 in the subcellular locations where Ab is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.  

3.147           Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport

Maier, V.H. and Gould G.W. Diabetologia, 43(10), 1273-1281 (2000)   Aimslhypothesis.  Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface.  It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance.  Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-Ll adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation.  Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment.   Methods  Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment, which probably represents GLUT4 storage vesicles (GSVs).   Results.  A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 ± 3.5 %) with only a modest decrease from the endosomal fraction (23 ± 2.6 %).  Long-term insulin treatment decreased cellular GLUT4 content by about 40 % and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation.  We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 ± 7 % decrease compared to untreated cells).   Conclusions/interpretation.  Such data argue that long-term insulin treatment results in the mistargeting of GLUT4 such that it no longer accesses the GSV compartment.  These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance.  

3.148           Assembly of myelin by association of proteolipid protein with cholesterol and  galactosylceramide-rich membrane domains

Simons, M., Kramer, E-M., Thiele, C., Stoffel, W. And Trotter, J.

1. Cell Biol., 151(1), 143-153 (2000)

  Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a lower-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF: Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycophingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.  

3.149           Intracellular events in the assembly of chylomicrons in rabbit enterocytes

Cartwright, I.J., Plonne, D. and Higgins, J.A.

1. Lipid Res., 41, 1728-1739 (2000)

  The aim of this study was to determine the intracellular events in chylomicron assembly in adult villus enterocytes. We have used novel methods for separation of the intracellular components of the secretory compartment [ rough and smooth endoplasmic reticulum (RER and SER), respectively) and Golgi], and their membrane and luminal components, from villus enterocytes isolated from rabbit small intestine. The steady state composition of the components of the secretory compartment and the intracellular pools of newly synthesized. Apolipoprotein B-48 (apoB-48) and triacylglycerol (TAG) was determined. The observations indicate that the SER is the main site of the TAG synthesis and of chylomicron assembly. Newly synthesized apoB-48 and TAG accumulate in the SER membrane and are transferred into the lumen in a microsomal triglyceride transfer protein-dependent step. In enterocytes isolated from chow-fed rabbits, in which fat absorption is relatively slow, transfer of apoB-48 and TAG from the SER membrane into the lumen appears to be rate limiting. In enterocytes from fat-fed rabbits, TAG accumulates into the lumen of the SER, suggesting that movement out of the SER lumen becomes rate limiting, when chylomicron secretion is markedly stimulated. In these cells, the cytosolic TAG also increased to 450 mg/g enterocytes, compared with 12 mg/g enterocytes from chow-fed rabbits, indicating that transfer of TAG from the SER membrane into the secretory pathway can become saturated, so that newly synthesized TAG moves into the cytosol.  

3.150           Brain plasmin enhances APP a-cleavage and Ab degradation and is reduced in Alzheimer’s disease brains

Ledesma, M.D., Da Silva, J.S., Crassaerts, K., Delacourte, A., De Strooper, B and Dotti, C.G. EMBO Reports, 11(61), 530-535 (2000)   The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the a-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain tissue from Alzheimer’s disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer’s disease.  

3.151           Cytosolic phospholipase A₂ regulates Golgi structure and modulates intracellular trafficking of membrane proteins

Choukroun, G.J. et al

1. Clin. Invest, 106, 983-993 (2000)

  The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane protein trafficking vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A₂ (cPLA₂) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA₂ in LLC-PK₁ kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na⁺-K⁺-ATPase a-subunit localization is markedly reduced in cells expressing cPLA₂, whereas the trafficking of a Cl ^-/ HCO₃^- anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA₂ results in dispersion of giantin and b-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA₂  is present in Golgi fractions from noninfected LLC-PK₁ cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA₂  indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA₂ activity. Thus cPLA₂ plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.  

3.152           Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus

Sinzger, C. et al

1. Gen. Virol., 81, 3021-3035 (2000)

  Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of the HCMV that determines this phenotype. The infection efficiency of various HCMV strains in EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late viral antigens. Adsorption and penetration were analyzed by radiolabeled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100-1000-fold lower when compared to endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression indicated the importance of initial replication events. Surprisingly, no interstrain differences were detected during virus entry. However, dramatic interstrain differences were found regarding the nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of viral DNA in the cell nucleus was 100-1000-fold lower in EC when compared to endotheliotropic strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of viral particles and viral IE antigen revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.  

3.153           B cell antigen receptor signaling occurs outside lipid rafts in immature B cells

Sproul, T.W., Malapati, S., Kim, J. and Pierce, S.

1. Immunol., 165, 6020-6023 (2000)

  B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic differences in the outcome of signaling in mature and immature B cells.  

3.154           Different properties of two isoforms of annexin XIII in MDCK cells

Lecat, S. et al

1. Cell Sci., 113, 2607-2618 (2000)

  Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.  

3.155           New and re-emerging diseases: A dedication to Norman D. Levine

Docampo, R. Parasitology Today, 16(8), 316, (2000)   No abstract  

3.156           Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates

Dunphy, J.T. et al Biochem. Biophys. Acta, 1485, 185-198 (2000)   Both enzymatic and autocatalytic mechanisms have been proposed to account for protein thioacylation (commonly known as palmitoylation). Acyl-CoA binding proteins (ACBP) strongly suppress non-enzymatic thioacylation of cysteinyl-containing peptides by long-chain acyl-CoAs. At physiological concentrations of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acylation is expected to be too slow to contribute significantly to thioacylation of signaling proteins in mammalian cells (Leventis et al., Biochemistry 36 (1997) 5546-5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characterized using G-protein a-subunits as a substrate (Dunphy et al., J. Biol. Chem., 271 (1996) 7154-7159), was capable of thioacylating short lipid-modified cysteinyl-containing peptides.The minimum requirements for substrate recognition were a free cysteins thiol adjacent to a hydrophobic lipid anchor, either myristate or farnesyl isoprenoid. PAT activity displayed specificity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhibited enzymatic thioacylation of a myristoylated peptide or G-protein a-subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl CoA in vitro and is likely to represent the predominant mechanism of thioacylation in vivo.  

3.157           Cytosolic Hsp70s are involved in the transport of aminopeptidase 1 from the cytoplasm into the vacuole

Satyanarayana, C., Schroder-Kohne, S., Craig, E.A., Schu, P.V. and Horst, M. FEBS Lett., 470, 232-238 (2000)   Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.  

3.158           Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay)

Shaw, J.P., Large, A.T., Chipman, J.K., Livingstone, D.R. and Peters, L.D. Marine Environmental Res., 50, 405-409 (2000)   Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Pot Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P< 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).  

3.159           Tyrosine mutants are capable of prodrug activation in transfected nonmelanotic cells

Simonova, M., Wall, A., Weissleder, R. and Bogdanov, A. Cancer Res., 60, 6656-6662 (2000)   Tyrosinase has been suggested as a prodrug-converting enzyme for the treatment of melanoma. We hypothesized that tyrosinase expression in transfected nonmelanotic cells can be used in a gene therapy paradigm of prodrug activation. To verify our hypothesis, we used the following tyrosinase variants: (a) a full-length human tyrosinase clone (T); (b) a mutant lacking the COOH-terminal cytoplasmic domain (TDC); (c) a mutant lacking the COOH-terminal transmembrane and cytoplasmic domains (TDTC); and (d) a fusion with the eight COOH-terminal amino acids of lysosome-associated membrane protein-1 (TL). Expression of mutant and wild-type tyrosinases was induced by transfection in nontumorigenic human cells of epithelial origin (293HEK, MCF-10A adenoma, and NHDF-Ad human dermal fibroblasts) as well as in tumour cells (9L gliocarcinoma, MCF7 adenocarcinoma and HT-1080 fibrosarcoma). When compared with the wild-type tyrosinase transfectants, truncated mutant expression resulted in higher mRNA levels that paralleled higher enzyme activity of the truncated mutants. Two model tyrosinase prodrugs, hydroxyphenyl-propanol (HPP) and N-acetyl-4-S-cysteaminylphenol (NAcSCAP) inhibited proliferation and caused cell death of transfected cells in a dose-dependent manner. Effects of prodrug treatment were compared for tumorigenic cells and their nontumorigenic counterparts. Two truncated mutants (TDC and TDTC) showed low endogenous cytotoxicity and efficiently suppressed proliferation and induced cytotoxicity in transfected tumor cells in the presence of NAcSCAP. Overall, these results indicate that the developed tyrosinase mutants hold promise as prodrug activation systems for tumoral gene therapy.  

3.160           GFRa –mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival

Tansey, M.G., Baloh, R.H., Milbrandt, J. and Johnson, Jr., E.M. Neuron, 25, 611-623 (2000)   The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a glycosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRa1-a4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRa 1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRa1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.  

3.161           Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer’s disease

Takahashi, M. et al Neuron, 28, 461-473 (2000)   Amyloid precursor protein (APP) generates the b-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer’s disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer’s disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer’s disease.  

3.162           Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-ras4B

Roy, M-O., Leventis, R. and Silvius, J.R. Biochemistry, 39, 8298-8307 (2000)   Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.  

3.163           An ever-expanding story of cyst formation

Gallagher, A.R., Obermüller, N., Cedzich, A., Gretz, N. and Witzgall, R. Cell Tissue Res., 300, 361-371 (2000)   Autosomal-dominant polycystic kidney disease represents one of the most common monogenetic human disorders. The cloning of the PKD1 and PKD2 genes, which are mutated in far more than 90% of the patients affected by this disease, has generated high hopes for a quick understanding of the pathogenesis of cyst formation. However, these expectations have not yet been fulfilled, since the function of both polycystin-1 and polycystin-2, the two proteins encoded by PKD1 and PKD2, still remains a puzzle. In this review, we will highlight some of the characteristics of polycystic kidney disease, briefly touch on polycystin-1, and then go on to describe recent results of experiments with polycystin-2, since the latter is the major focus of our work. We will discuss new evidence which suggests that autosomal-dominant polycystic kidney disease actually behaves recessively on a cellular level. Finally, a model will be presented that tries to explain the available data.  

3.164           Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

Fengsrud, M., Erichsen, E.S., Berg, T.O., Raiborg, C. And Seglen, P.O. Eur. J. Cell Biol., 79, 871-882 (2000)   The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/um², whereas isolated lysosomes had about 2000 particles/um²). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/um²), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearance of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi-or multilammelar autophagic vacuoles appeared to engage in such fusions.          

3.165           The phagacytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

Seres, T., Knickelbein, R.G., Warshaw, J.B. and Johnston, Jr, R.B.

1. Immunol., 165, 3333-3340 (2000)

  During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [³⁵S]glutathione ([³⁵S]GSH) after 20–30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H₂O₂), and micromolar H₂O₂ stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H₂O₂ and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by -glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a K_m (8.5 µM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H₂O₂ during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.  

3.166           Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Chan, D., Lin, J. and Raffaniello, R.D.

1. Cell. Physiol., 185(3), 339-347 (2000)

  Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na2CO3, indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.  

3.167           Mutation of conserved aspartates affect maturation of presenilin 1 and presenilin 2 complexes

Yu, G., Chen, F., Nishimura, M., Steiner, H., Tandon, A., Kawarai, T., Arawaka, S., Supala, A., Song, Y-Q., Rogaeva, E., Holmes, E., Zhang, D.M., Milman, P., Fraser, P., Haass, C. and  St. George-Hyslop, P. Acta Neurol Scand., Suppl. 176, 6-11 (2000)   Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments (NTF/CTF). Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of APP and Notch. We show that aspartate-mutant holoprotein presenilins are not incorporated into the high molecular weight, NTF/CTF-containing complexes. Aspartate-mutant presenilin holoproteins also preclude entry of endogenous wild-type PS1/PS2 into the high molecular weight complexes, but do not affect the incorporation of wild-type holoproteins into lower molecular weight holoprotein complexes. These data suggest that the loss-of-function aspartate-mutants cause altered PS complex maturation, and argue that the functional presenilin moieties are contained in the high molecular weight presenilin NTF/CTF-containing complexes.  

3.168           Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a  putative lipase

Teter, S.A. et al

1. Biol. Chem., 276(1), 2083-2087 (2001)

  The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions.  Vacuolar hydrolases are key proteins in this process.  In Saccharomyces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy.  Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions.  Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole.  As a result, these pathways share a common terminal step, degradation of subvacuolar vesicles.  We have identified a protein, Cvt17, which is essential for this membrane lytic event.  Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases.  The active-site serine of this motif is required for sub-vacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.  

3.169           An ephrinA-dependent signaling pathway controls integrin function and is linked to the tyrosine phosphorylation of a 120 kDa protein

Huai, J.and Drescher, U.

1. Biol. Chem., 276(9), 6689-6694 (2001)

  The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection.  Here, glycosylphoaphatidylinositol-anchored A-ephrins are not only expressed in the tectum, but also on retinal axons, raising the possibility that they function in this context as receptors.  We now show that activation of ephrinA2 or ephrinA5 by one of their receptors, ephA3, results in a b1-integrin dependent increased adhesion of ephrinA-expressing cells to laminin.  In the search for an ephrinA-dependent signaling pathway controlling integrin activation, we identified a 120 kDa raft membrane protein which is tyrosine phosphorylated specifically after ephrinA activation.  Tyrosine phosphorylation of this protein is not seen after stimulating ephrinA2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor or fetal calf serum containing a large set of different growth factors.  The role of p120 as a mediator of an ephrinA - integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of pl20 correlates with an abolishment of the b1-dependent cell adhesion.  

3.170           The synaptic vesicle protein, cysteine-string, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4

Chamberlain, L.H. et al

1. Cell Sci., 114(2), 445-455 (2001)

  Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localized to specialized intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterize proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocytes plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Cps1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with plasma membrane.  

3.171           Pro-caspase-8 is pre-dominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

Qin, Z-H. et al

1. Biol. Chem., 276(11), 8079-8086 (2001)

  The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was co-localized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in the nucleus. Pro-caspase-8 was found in the intermembrane space, inner membrane and matrix of mitochondria after limited digestion of mitochondrial fractions and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside (Atr), which opens the mitochondrial permeability transition pore. Release was blocked by the mitochondrial permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 hr to the apoptotic inducer, tumor necrosis factor-a (TNF-a), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8a cleavage products, in the cytoplasm of the TNF-alpha treated striatal cells. CsA blocked the TNF-a-induced release of pro-caspase 8, but not cytochrome c. Internucleosomal DNA fragmentation started at 6 hr and peaked at 12 hr after TNF-a treatment. These results suggest that pro-caaspase-8 is predominately localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.  

3.172           Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth  factor receptor

Chin, L-S., Raynor, M.C., Wei, X., Chen, H-Q. and  Li, L.

1. Biol. Chem., 276(10), 7069-7078 (2001)

  Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p, however the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognize the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a ~550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using depletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.  

3.173           Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes

Shisheva, A., Rusin, B., Ikonomov, O.C., DeMarco, C. and Sbrissa, D.

1. Biol. Chem., 276(15), 11859-11869 (2001)

  The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P₂, thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve product’s formation, we have examined the localization of PIKfyve protein vs. enzymatic activity, and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol (~76%), internal structures (LDM, composed of recycling endosomes, GLUT4 storage compartment, Golgi and cytoskeletal elements) (~20%), and plasma membrane (~4%), expressed enzymatically active PIKfyve. While presence of FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve’s role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.  

3.174           Active (9.6S) and inactive (21S) oligomers of NHE3 in distinct microdomains of the  renal brush border

Biemesderfer, D., DeGray, B. and Aronson, P.S.

1. Biol. Chem., 276(13), 10161-10167 (2001)

  We have previously shown that Na⁺-H⁺ exchanger isoform NHE3 exists as both 9.6 S and 21 S (megalin-associated) oligomers in the renal brush border (Biesmesderfer et al., JBC 274, 17518, 1999). To characterize the oligomeric forms of the renal brush border Na⁺-H⁺ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a non-microvillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter co-localized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 co-precipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S-value of 9.6 while the S-value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na⁺ gradient caused generation of an inside-acid pH gradient in the microvilli, indicating Na⁺-H⁺ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states; a 9.6 S active form present in microvilli, and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na⁺-H⁺ exchange activity may be mediated by shifting the distribution between these forms of NHE3.  

3.175           Membrane lipid rafts are necessary for the maintenance of the a7 nicotinic  acetylcholine receptor in somatic spines of ciliary neurons

Bruses, J.L., Chauvet, N. and Rutishauser, U.

1. Neurosci., 21(2), 504-512 (2001)

  Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission.  The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface.  The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable a7-subunit nicotinic acetylcholine receptor (a7nAChR) clusters found in somatic spines of ciliary neurons.  Lipid rafts and a7nAChR become progressively colocalized within somatic spines during synaptogenesis.  To determine whether these rafts are required for the maintenance of a7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-b-cyclodextrin.  This treatment caused the dispersion of lipid rafts and the redistribution of a7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering.  However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites.  To assess whether interaction between rafts and a7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX.  The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin.  Under these conditions, large patches of CTX persisted and were colocalized with a7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton.  Moreover, it was found that the a7nAChR is resistant to detergent extraction at 4⁰C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.  

3.176           Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex

Lukong, K.E. et al

1. Biol. Chem., 276, 17286-17290 (2001)

  Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates.  The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type 1) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II).  We have analyzed the effect of missense mutations, G68V, Sl82G, G227R, F26OY, L270F, A298V, G328S and L363P, identified in the sialidosis type I and sialidosis type II patients on the activity, stability and intracellular distribution of the sialidase.  We found that 3 mutations, F260Y, L270F and A298V which are clustered in the same region on the surface of sialidase molecule dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein.  We suggested that this region might be involved in the sialidase binding with lysosomal cathepsin A and/or b-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity.  Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.  

3.177           Mammalian sprouty-1 and –2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells

Impagnatiello, M-A. et al

1. Cell Biol., 152(5), 1087-1098 (2001)

  Growth factor-induced signaling by receptor tyrosine-kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subse-quently, four mammalian Sprouty homologues (Spry 1-4) have been identified. Here, we report for funct-ional characterization of two of them, Spry-1 and –2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and different-iation by repressing pathways leading to p42/44 mitogen activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells were also inhibited by Spry-1 and –2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and –2 reveal that both Spry-1 and –2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in an RTK-specific fashion.  

3.178           Cytosolic phospholipase A₂-a associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells

Liu, J., Takano, T., Papillon, J., Khadir, A. and Cybulsky, A.V. Biochem. J., 353, 79-90 (2001)   Eicosanoids mediate complement-dependent glolmerular epi­thelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A₂ (cPLA₂) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA₂ with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca²⁺ -free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA₂, and organelles was analyzed by immunoblotting with antibodies against cPLA₂ and organelle markers, or by enzyme assay. In cells incubated with or without the Ca²⁺ ionophore ionomycin plus PMA, cPLA₂ co-localized with plasma mem­brane, endoplasmic reticulum and nuclei, but not with mito­chondria or Golgi. A greater amount of cPLA₂ was associa­ted with membranes in stimulated cells, but membrane-associated cPLA₂ was readily detectable under resting conditions. The pattern of association of cPLA₂ with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA₂ protein. To determine the functional role of membrane association of cPLA₂, phospholipids were labeled with [³H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radio­activity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA₂ is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.  

3.179           Raft-partitioning of the ubiquitin ligases CbI and Nedd4 upon IgE-triggered cell signaling

Lafont, F. and Simons, K. Proc. Natl. Acad. Sci., USA., 98; 3180-3184 (2001).   The high affinity receptor for IgE, FceRI on mast calls and basophils plays an essential role in immunological defense. Upon multivalent antigen binding,  FceRI becomes phoshorylated by the protein-­tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FceRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FceRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basolukemia cells for the presence of ubiquitinated FceRI in clustered rafts upon receptor activation. Moreover, we demon­strated that the ubiquitin ligases CbI and Nedd4 co-localize with FceRI patches and showed that both ligases become assoc-iated with lipid rafts after activation of IgE signaling. Because CbI is known to interact with the FceRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.  

3.180           Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria

Li, W-P et al

1. Cell Sci., 114(7), 1397-1408 (2001)

  In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate cevaolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.  

3.181           Glycolipid antigen processing for presentation by CD1d molecules

Prigozy, T.I. et al Science, 291, 664-667 (2001)   The requirement for processing glycolipid antigens in T cell recognition was examined with mouse CD1d-mediated responses to glycosphingolipids (GSLs). Although some disaccharide GSL antigens can be recognized without processing, the responses to three other antigens, including the disaccharide GSL Gal(a1®2)GalCer (Gal, galactose; GalCer, galactosylceramide), required removal of the terminal sugars to permit interaction with the T cell receptor. A lysosomal enzyme, a-galactosidase A, was responsible for the processing of Gal(a1®2)GalCer to generate the antigenic monosaccharide epitope. These data demonstrate a carbohydrate antigen processing system analogous to that used for peptides and an ability of T cells to recognize processed fragments of complex glycolipids.  

3.182           Segregation of heterotrimeric G proteins in cell surface microdomains

Oh, P. and Schnitzer, J.E. Mol. Biol. Cell, 12, 685-698 (2001)   Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinase may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G_i, G_q, G_s, and G_bg) target discrete cell surface microdomains. G_q specifically concentrates in caveolae, whereas G_i and G_s concentrate much more in lipid rafts marked by GPI-anchored proteins (5’ nucleotidase and folate receptor). G_q, apparently without G_bg subunits, stably associates with plasmalemmal and cytosolic caveolin. G_i and G_s interact with G_bg subunits but not caveolin.G_i and G_s unlike G_q, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G_q preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G_q, G_i, and G_s all concentrate in lipid rafts as a complex with G_bg. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G_q-caveolin complex without G_bg for trafficking, signaling, and mechanotransduction are discussed.  

3.183           Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

Lusa, S., et al

1. Cell Sci., 114(10), 1893-1900 (2001)

  In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signaling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [³H]cholesterol released from [³H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.  

3.184           The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase

Legare, D., et al

1. Biol. Chem., 276(28), 26301-26307 (2001)

  The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to gluthathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein (GFP) were fused at the COOH-terminus of PGPA. Immunofluorescence, confocal and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PG-PAHA transfectants was performed to characterize further this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmoles/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transport thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.  

3.185           Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole

Kim, J. et al

1. Cell Biol., 153(2), 381-396 (2001)

  Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.   We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.  

3.186           Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the a-secretase ADAM 10

Kojro, E., Gimpl, G., Lammich, S., Marz, W. and Fahrenholz, F. Proc. Natl. Acad. Sci., 98(10), 5815-5820 (2001)   Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer’s disease. In the present report, we identify the a-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neutral cell lines with either the cholesterol-extracting agent methyl-b-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastin resulted in a drastic increase of secreted a-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of a-secretase activity resulted in a decreased secretion of Ab peptides. Several mechanisms were elucidated as being the basis of enhanced a-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-b-cyclodextrin; treatment with lovastatin resulted in higher expression of the a-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic a-secretase pathway and the formation of neuroprotective a-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer’s disease.  

3.187           Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Frenzel, K.E. and Falls, D.L.

1. Neurochem., 77, 1-12 (2001)

  Neuregulin–1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synatosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140-kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-b1a or the III-b1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-b1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.  

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