Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.     

The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.     

Apolipoprotein A-V N-terminal Domain Lipid Interaction Properties in Vitro Explain the Hypertriglyceridemic Phenotype Associated with Natural Truncation Mutants

The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1–146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1–146) forms reconstituted high density lipoproteins 15–17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1–146) yielded an α-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1–146) α-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1–146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1–146) α-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1–146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1–146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects.     

Altered apolipoprotein A-V expression during the acute phase response is independent of plasma triglyceride levels in mice and humans

Plasma triglyceride (TG) levels are altered during the acute phase response (APR). Plasma levels of the recently discovered apolipoprotein A-V (apoA-V) are inversely associated with plasma TG. The aim of this study was to investigate the change of apoA-V plasma levels and hepatic apoA-V expression during the APR in relation to plasma TG. During human APR plasma apoA-V was decreased as were plasma TG (each P<0.01). Also early in the course of the murine APR plasma apoA-V levels and hepatic apoA-V expression were decreased and changed in the same direction as plasma TG. Treatment of HepG2 cells with TNF-alpha and IL-1beta decreased apoA-V mRNA levels early by 42% and 55%, respectively (each P<0.001). However, in promoter/reporter assays the human apoA-V promoter was unresponsive to proinflammatory cytokines. Instead, we demonstrate that a significant decrease in apoA-V mRNA stability in response to treatment with TNF-alpha and IL-1beta is the underlying basis of decreased apoA-V expression during the APR (P<0.05). These data demonstrate that (i) apoA-V expression decreases early during the APR due to changes in mRNA stability, and (ii) during the APR apoA-V is not inversely related to plasma TG levels in mice and humans, thereby identifying a relevant pathophysiological setting, in which the previously reported close inverse association between these parameters does not hold true.     

Evidence for a complex relationship between apoA-V and apoC-III in patients with severe hypertriglyceridemia

The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8-78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG.     

A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration

Apolipoprotein A-V (apoA-V) is a recently discovered apolipoprotein that appears to have a role in plasma triglyceride (TG) transport. We have developed an ELISA for apoA-V using monoclonal antibodies that has a lower limit of detection of 0.3 ng/ml and linearity up to 20 ng/ml. The ELISA was then used to quantify plasma apoA-V in 196 healthy subjects and 106 patients with insulin-resistant diabetes mellitus. In the healthy subjects, total apoA-V concentration was 179.2 +/- 74.8 ng/ml, and it was greater in females than in males (P < 0.005). It was correlated positively with the plasma HDL cholesterol (r = 0.32, P < 0.0001), apoA-I (r = 0.27, P = 0.0001), and apoE (r = 0.18, P = 0.011) concentrations and negatively with plasma TG concentration (r = -0.22, P = 0.021). In relation to single nucleotide polymorphism 3 (-1131C/T) of the apoA-V gene, apoA-V concentration was higher in the T/T type than in the C/C type (P < 0.01). Plasma TG concentration was lower in the T/T type than in the C/C or C/T type (P < 0.05). ApoA-V concentration was lower in the diabetic patients (69.4 +/- 44.3 ng/ml; P < 0.01) than in the healthy controls.     

Apolipoprotein A-V dependent modulation of plasma triacylglycerol: a puzzlement

The discovery of apolipoprotein A-V (apoA-V) in 2001 has raised a number of intriguing questions about its role in lipid transport and triglyceride (TG) homeostasis. Genome wide association studies (GWAS) have consistently identified APOA5 as a contributor to plasma TG levels. Single nucleotide polymorphisms (SNP) within the APOA5 gene locus have been shown to correlate with elevated plasma TG. Furthermore, transgenic and knockout mouse models support the view that apoA-V plays a critical role in maintenance of plasma TG levels. The present review describes recent concepts pertaining to apoA-V SNP analysis and their association with elevated plasma TG. The interaction of apoA-V with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is discussed relative to its postulated role in TG-rich lipoprotein catabolism. The potential role of intracellular apoA-V in regulation of TG homeostasis, as a function of its ability to associate with cytosolic lipid droplets, is reviewed. While some answers are emerging, numerous mysteries remain with regard to this low abundance, yet potent, modulator of TG homeostasis. Given the strong correlation between elevated plasma TG and heart disease, there is great scientific and public interest in deciphering the numerous biological riddles presented by apoA-V. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM β-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in T(m), displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T(m) increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information.     

Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice

Transgenic (Tg) mice that overexpress the human apolipoprotein A-V gene (APOA5) yet lack an endogenous mouse apoa5 gene (APOA5 Tg mice) were generated. Subsequently, the effect of human apoA-V expression on plasma triglyceride (TG) concentration and lipoprotein and apolipoprotein distribution was determined and compared with that in mice deficient in apoA-V (apoa5(-/-) mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 +/- 7.7 nmol/dl), whereas VLDL in apoa5(-/-) mice was 18- fold higher (467 +/- 152 nmol/dl). SDS-PAGE analysis of the d < 1.063 g/ml plasma fraction revealed that the apoB-100/apoB-48 ratio was 14-fold higher in APOA5 Tg versus apoa5(-/-) mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5(-/-) mice. It is anticipated that a reduction in apoB-100/apoB-48 ratio as well as that for apoE/apoB would impair the uptake of VLDL and remnants in apoa5(-/-) mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 +/- 2.9 microg/ml, which is approximately 50- to 100-fold higher than that reported for normolipidemic humans. ApoA-V was predominantly associated with HDL but was rapidly and efficiently redistributed to apoA- V-deficient VLDL upon incubation. Consistent with findings reported for human subjects, apoA-V concentration was positively correlated with TG levels in normolipidemic APOA5 Tg mice. It is conceivable that, in a situation in which apoA-V is chronically overexpressed, complex interactions among factors regulating TG homeostasis may result in a positive correlation of apoA-V with TG concentrations.     

Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis

To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% alpha-helix, 33% beta-sheet, 16% beta-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 degrees C. Upon being cooled to ambient temperature from 85 degrees C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15-20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 +/- 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.     

Avian apolipoprotein A-V binds to LDL receptor gene family members

Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V.     

Biogenesis of apolipoprotein A-V and its impact on VLDL triglyceride secretion

Apolipoprotein A-V (apoA-V) is a potent regulator of intravascular triglyceride (TG) metabolism, yet its plasma concentration is very low compared with that of other apolipoproteins. To examine the basis for its low plasma concentration, the secretion efficiency of apoA-V was measured in stably transfected McA-RH7777 rat hepatoma cells. Pulse-chase experiments revealed that only ～20% of newly synthesized apoA-V is secreted into culture medium within 3 h postsynthesis and that ～65% undergoes presecretory turnover; similar results were obtained with transfected nonhepatic Chinese hamster ovary cells. ApoA-V secreted by McA-RH7777 cells was not associated with cell surface heparin-competable binding sites. When stably transfected McA-RH7777 cells were treated with oleic acid, the resulting increase in TG synthesis caused a reduction in apoA-V secretion, a reciprocal increase in cell-associated apoA-V, and movement of apoA-V onto cytosolic lipid droplets. In a stably transfected doxycycline-inducible McA-RH7777 cell line, apoA-V expression inhibited TG secretion by ～50%, increased cellular TG, and reduced Z-average VLDL(1) particle diameter from 81 to 67 nm; however, no impact on apoB secretion was observed. These data demonstrate that apoA-V inefficiently traffics within the secretory pathway, that its intracellular itinerary can be regulated by changes in cellular TG accumulation, and that apoA-V synthesis can modulate VLDL TG mobilization and secretion.     

Apolipoprotein A-V, triglycerides and risk of coronary artery disease: the prospective Epic-Norfolk Population Study

In mouse models, apolipoprotein A-V (apoA-V) exhibits triglyceride (TG)-lowering effects. We investigated the apoA-V/TG relationship and the association of apoA-V with coronary artery disease (CAD) risk by determining serum apoA-V levels and genotypes in a nested case-control (n = 1,034/2,031) study. Both univariate and multivariate apoA-V levels showed no association with future CAD (P = 0.4 and 0.5, respectively). Unexpectedly, there was a significant positive correlation between serum apoA-V and TG in men and women (r = 0.36 and 0.28, respectively, P < 0.001 each) but a negative correlation between apoA-V and LPL mass (r = -0.14 and -0.12 for men and women respectively, P < 0.001 each). The frequency of the c.56C>G polymorphism did not differ between cases and controls despite significant positive association of c.56G with both apoA-V and TG levels. For -1131T>C, the minor allele was significantly associated with lower apoA-V yet higher TG levels and was overrepresented in cases (P = 0.047). The association of -1131T>C with CAD risk, however, was independent of apoA-V levels and likely acts through linkage disequilibrium with APOC3 variants. The positive correlation of apoA-V levels with TG levels, negative correlation with LPL levels, and lack of association with CAD risk highlight the need for further human studies to clarify the role of apoA-V.     

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation

Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V (∼ 80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by ∼ 25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting that apoA-V influences intrahepatic TG levels.     

Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.     

Influence of apoA-V gene variants on postprandial triglyceride metabolism: impact of gender

Although apolipoprotein A-V (apoA-V) polymorphisms have been consistently associated with fasting triglyceride (TG) levels, their impact on postprandial lipemia remains relatively unknown. In this study, we investigate the impact of two common apoA-V polymorphisms (-1131 T>C and S19W) and apoA-V haplotypes on fasting and postprandial lipid metabolism in adults in the United Kingdom (n = 259). Compared with the wild-type TT, apoA-V -1131 TC heterozygotes had 15% (P = 0.057) and 21% (P = 0.002) higher fasting TG and postprandial TG area under the curve (AUC), respectively. Significant (P = 0.038) and nearly significant (P = 0.057) gender x genotype interactions were observed for fasting TG and TG AUC, with a greater impact of genotype in males. Lower HDL-cholesterol was associated with the rare TC genotype (P = 0.047). Significant linkage disequilibrium was found between the apoA-V -1131 T>C and the apoC-III 3238 C>G variants, with univariate analysis indicating an impact of this apoC-III single nucleotide polymorphism (SNP) on TG AUC (P = 0.015). However, in linear regression analysis, a significant independent association with TG AUC (P = 0.007) was only evident for the apoA-V -1131 T>C SNP, indicating a greater relative importance of the apoA-V genotype.     

Apolipoprotein A-V-heparin interactions: implications for plasma lipoprotein metabolism

Transgenic and gene disruption experiments in mice have revealed that apolipoprotein (apo) A-V is a potent regulator of plasma triglyceride (TG) levels. To investigate the molecular basis of apoA-V function, the ability of isolated recombinant apoA-V to modulate lipoprotein lipase (LPL) activity was examined in vitro. With three distinct lipid substrate particles, including very low-density lipoprotein (VLDL), a TG/phospholipid emulsion, or dimyristoylphosphatidylcholine liposomes, apoA-V had little effect on LPL activity. In the absence or presence apolipoprotein C-II, apoA-V marginally inhibited LPL activity. On the other hand, apoA-V-dimyristoylphosphatidylcholine disc particles bound to heparin-Sepharose and were specifically eluted upon application of a linear gradient of NaCl. The interaction of apoA-V with sulfated glycosaminoglycans was further studied by surface plasmon resonance spectroscopy. ApoA-V showed strong binding to heparin-coated chips, and binding was competed by free heparin. ApoA-V enrichment enhanced binding of apoC-II-deficient chylomicrons and VLDL to heparin-coated chips. When LPL was first bound to the heparin-coated chip, apoA-V-enriched chylomicrons showed binding. Finally, human pre- and post-heparin plasma samples were subjected to immunoblot analysis with anti-apoA-V IgG. No differences in the amount of apoA-V present were detected. Taken together, the results show that apoA-V lipid complexes bind heparin and, when present on TG-rich lipoprotein particles, may promote their association with cell surface heparan sulfate proteoglycans. Through such interactions, apoA-V may indirectly affect LPL activity, possibly explaining its inverse correlation with plasma TG levels.     

The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer.

The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides. Hfq particularly affects the translation and the stability of several RNAs. In an earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology. This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein. Hfq forms a beta-sheet ring-shaped hexamer. As our previous study predicted a large number of alternative conformations for the C-terminal region, we have determined whether the last 19 C-terminal residues are necessary for protein function. We find that the C-terminal truncated protein is fully capable of binding a polyadenylated RNA (K(d) of 120 pm vs. 50 pm for full-length Hfq). This result shows that the functional core of E. coli Hfq resides in residues 1-70 and confirms previous genetic studies. Using equilibrium unfolding studies, however, we find that full-length Hfq is 1.8 kcal x mol(-1) more stable than its truncated variant. Electron microscopy analysis of both truncated and full-length proteins indicates a structural rearrangement between the subunits upon truncation. This conformational change is coupled to a reduction in beta-strand content, as determined by Fourier transform infra-red. On the basis of these results, we propose that the C-terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure. The origin of this C-terminal domain is also discussed.     

Inhomogeneous stability of bacteriorhodopsin in purple membrane against photobleaching at high temperature

Heterogeneity in the state of bacteriorhodopsin in purple membrane was studied through temperature jump experiments carried out in darkness and under illumination with visible light. The thermal denaturation, the irreversible component of spectral change at high temperature, had two decay components, suggesting that bacteriorhodopsin in purple membrane has heterogeneous stability. The temperature dependence of kinetic parameters under illumination revealed that the fast-decay component gradually increased at above 60 degrees C, indicating that the proportion of unstable bacteriorhodopsin increased. Significant change in the visible circular dichroism (CD) spectra was observed in darkness in the same temperature range as the increase of the fast-decay component under illumination. Denaturation experiments for C-terminal-cleaved bacteriorhodopsin showed that the C-terminal segment had some effect on the structural stability of bacteriorhodopsin under illumination. Dynamic and static models of the inhomogeneous stability of bacteriorhodopsin in purple membrane are discussed on the basis of the results of the denaturation kinetics and the visible CD spectra.