Thiol-bearing synthetic peptides retain the antioxidant activity of apolipoproteinA-I(Milano)

Apolipoprotein(apo)A-I(Milano) (R173C) and apoA-I(Paris) (R151C) are rare cysteine variants of wild-type (WT) apoA-I that possess novel antioxidant properties on phospholipid surfaces. Yet, the two variants differ in their ability to inhibit lipid peroxidation. In this study, we used synthetic peptides (18mers) to investigate the structural basis for the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris). A peptide (aa 167-R173C-184) based on the amphipathic alpha helix harboring the R173C mutation inhibited superoxide anion-mediated oxidation of phospholipid in a dose-dependent manner, but it failed to directly quench superoxide anions in aqueous solution, indicating that the peptide acted at the level of phospholipid to inhibit lipid peroxidation just like the full-length cysteine variant. Peptide 145-R151C-162 based on the helical segment containing R151C exhibited the same capacity as peptide 167-R173C-184 to inhibit lipid peroxidation. Thus, the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris) was not governed by the primary amino acid sequence of their individual amphipathic alpha helices, rather contextual constraints within the full-length variants set the difference in antioxidant activity. Cysteine-free peptides were weak inhibitors of lipid peroxidation. These results suggest that thiol-bearing helical peptides based on apoA-I(Milano) may be useful to combat inflammatory related diseases.     

Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers

ApoA-I(R160L)Oslo and apoA-I(P165R) are naturally occurring apolipoprotein (apo) A-I variants that are associated with low HDL-cholesterol in heterozygous carriers. We characterized the capacity of these variants to bind lipid, to activate lecithin:cholesterol acyltransferase (LCAT), and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. During cholate dialysis, normal apoA-I and both variants associated completely with dipalmitoylphosphatidylcholine (DPPC) and formed rLpA-I of identical size. However, both apoA-I(P165R) and apoA-I(R160L)Oslo showed a reduced capacity to clear a turbid emulsion of dimyristoylphosphatidylcholine (DMPC). Compared to normal apoA-I, the LCAT-cofactor activity of apoA-I(P165R) and apoA-I(R160L)Oslo as defined by the ratio of Vmax to appKm was reduced significantly by 62% and 29%, respectively (here and throughout the text, the apparent Km is given as Michaelis-Menten kinetics do not take particle binding into account and therefore would result in errors with an interfacial enzyme such as LCAT; Vmax estimates are not affected by this error). ApoA-I/DPPC complexes induced biphasic cholesterol efflux from SMCs with a fast and a slow efflux component. Compared to rLpA-I reconstituted with wild type apoA-I, rLpA-I with apoA-I(P165R) or apoA-I(R160L)Oslo were significantly less effective in promoting cholesterol efflux from SMCs in incubations of 10 min duration but equally effective in incubations of 6 h duration. Lipid-free apoA-I did not induce efflux of biosynthetic cholesterol from SMCs but induced hydrolysis of cholesteryl esters and cholesterol efflux from acetyl-LDL-loaded mouse peritoneal macrophages. In the lipid-free form, both apoA-I variants promoted normal cholesterol efflux from murine peritoneal macrophages.We conclude that amino acid residues arginine 160 and proline 165 of apoA-I contribute to the formation of a domain that is very important for initial lipid binding and contributes to LCAT-activation and promotion of initial cholesterol efflux but not to the stabilization of preformed rLpA-I.     

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties

Apolipoprotein A-I(Milano) (A-I(M)) (R173C), a natural mutant of human apolipoprotein A-I (apoA-I), and five other cysteine variants of apoA-I at residues 52 (S52C), 74 (N74C), 107 (K107C), 129 (G129C), and 195 (K195C) were generated. Cysteine residues were incorporated in each of the various helices at the same helical wheel position as for the substitution in A-I(M). The secondary structural properties of the monomeric mutants, their abilities to bind lipid and to promote cholesterol efflux from THP-1 macrophages, and the possibility of antiperoxidation were investigated. Results showed that the alpha helical contents of all of the cysteine mutants were similar to that of wild-type apoA-I (wtapoA-I). The cysteine variant of A-I(M) at residue 173 [A-I(M)(R173C)] exhibited weakened structural stability, whereas A-I(G129C) a more stable structure than wtapoA-I. A-I(G129C) and A-I(K195C) exhibited significantly impaired capabilities to bind lipid compared with wtapoA-I. A-I(K107C) possessed a higher capacity to promote cholesterol efflux from macrophages than wtapoA-I, and A-I(M)(R173C) and A-I(K195C) exhibited an impaired efflux capability. Neither A-I(M)(R173C) nor any other cysteine mutant could resist oxidation against lipoxygenase. In summary, in spite of the similar mutant position on the helix, these variants exhibited different structural features or biological activities, suggesting the potential influence of the local environment of mutations on the whole polypeptide chain.     

Structural and functional basis for increased HDL-cholesterol levels due to the naturally occurring V19L mutation in human apolipoprotein A-I

Several hereditary point mutations in human apolipoprotein A-I (apoA-I) have been associated with low HDL-cholesterol levels and/or increased coronary artery disease (CAD) risk. However, one apoA-I mutation, the V19L, recently identified in Icelanders, has been associated with increased HDL-cholesterol levels and decreased CAD risk. In an effort to gain mechanistic insight linking the presence of this mutation in apoA-I with the increase of HDL-cholesterol levels we evaluated the effect of V19L mutation on the conformational integrity and functional properties of apoA-I in lipid-free and lipidated form. ApoA-I[V19L] was found to be thermodynamically destabilized in lipid-free form and displays an increased capacity to associate with phospholipids compared to WT apoA-I. When associated to reconstituted HDL (rHDL), apoA-I[V19L] was more thermodynamically stabilized than WT apoA-I. ApoA-I[V19L] displayed normal capacity to promote ABCA1-mediated cholesterol efflux and to activate the enzyme LCAT, in lipid-free and rHDL-associated forms, respectively. Additionally, rHDL-associated apoA-I[V19L] showed normal capacity to promote ABCG1-mediated cholesterol efflux, but 45% increased capacity to promote SR-BI-mediated cholesterol efflux, while the SR-BI-mediated HDL-lipid uptake was normal. Overall, our findings show that the apoA-I V19L mutation does not affect the first steps of HDL biogenesis pathway. However, the increased capacity of apoA-I[V19L] to associate with phospholipids, in combination with the enhanced thermodynamic stability of lipoprotein-associated apoA-I[V19L] and increased capacity of apoA-I[V19L]-containing lipoprotein particles to accept additional cholesterol by SR-BI could account for the increased HDL-cholesterol levels observed in human carriers of the mutation.     

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.     

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.     

Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan

Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with ΔE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the ΔE235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal α-helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal α-helical structure by the ΔE235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/ΔE235 peptide showed significantly decreased ability to form α-helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form α-helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands.     

Human apolipoprotein A-I and A-I mimetic peptides: potential for atherosclerosis reversal

PURPOSE OF REVIEW: Recent publications related to the potential use of apolipoprotein (apo)A-I and apoA-I mimetic peptides in the treatment of atherosclerosis are reviewed. RECENT FINDINGS: A preliminary report indicating that infusion of apoA-IMilano into humans once weekly for 5 weeks caused a significant decrease in coronary artery atheroma volume has sparked great interest in the potential therapeutic use of apoA-I. Recent studies have revealed that HDL quality (e.g. HDL apolipoprotein and lipid content, including oxidized lipids, particle size and electrophoretic mobility, associated enzymatic activities, inflammatory/anti-inflammatory properties, and ability to promote cholesterol efflux) may be more important than HDL-cholesterol levels. Therefore, when developing new strategies to raise HDL-cholesterol concentrations by interfering with HDL metabolism, one must consider the quality of the resulting HDL. In animal models, raising HDL-cholesterol levels by administering oral phospholipids improved both the quantity and quality of HDL and was associated with lesion regression. An apoA-I mimetic peptide, namely 4F synthesized from D-amino acids (D-4F), administered orally to mice did not raise HDL-cholesterol concentrations but promoted the formation of pre-beta HDL containing increased paraoxonase activity, resulting in significant improvements in HDL's anti-inflammatory properties and ability to promote cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol efflux from macrophages in vivo. SUMMARY: The quality of HDL may be more important than HDL-cholesterol levels. ApoA-I and apoA-I mimetic peptides appear to have significant therapeutic potential in atherosclerosis.     

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ?K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ?K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (?K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ?K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ?K107 or ?K226.     

Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.     

Synthesis and secretion of apolipoprotein A-I by chick skin.

Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin.     

Crystal structure of C-terminal truncated apolipoprotein A-I reveals the assembly of high density lipoprotein (HDL) by dimerization

Apolipoprotein A-I (apoA-I) plays important structural and functional roles in plasma high density lipoprotein (HDL) that is responsible for reverse cholesterol transport. However, a molecular understanding of HDL assembly and function remains enigmatic. The 2.2-? crystal structure of Δ(185-243)apoA-I reported here shows that it forms a half-circle dimer. The backbone of the dimer consists of two elongated antiparallel proline-kinked helices (five AB tandem repeats). The N-terminal domain of each molecule forms a four-helix bundle with the helical C-terminal region of the symmetry-related partner. The central region forms a flexible domain with two antiparallel helices connecting the bundles at each end. The two-domain dimer structure based on helical repeats suggests the role of apoA-I in the formation of discoidal HDL particles. Furthermore, the structure suggests the possible interaction with lecithin-cholesterol acyltransferase and may shed light on the molecular details of the effect of the Milano, Paris, and Fin mutations.     

Oxidation of apolipoprotein A-I by myeloperoxidase impairs the initial interactions with ABCA1 required for signaling and cholesterol export

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.     

Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice

Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.     

Enhanced binding of apolipoprotein A-I variants associated with hypertriglyceridemia to triglyceride-rich particles

Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains largely unknown. It was shown that severe HTG can be induced in mice by overexpression of wild-type (WT) apolipoprotein E (apoE) or specific apoA-I mutants. Certain mutations in apoE4 were found to affect plasma triglyceride (TG) levels in mice overexpressing the protein. HTG appeared to positively correlate with the ability of the apoE4 variants to bind to TG-rich particles, protein destabilization, and the exposure of protein hydrophobic surface in solution. Here, we propose that the apoA-I mutations that cause HTG may also lead to changes in the conformation and stability that promote binding of apoA-I to TG-rich lipoproteins. To test this hypothesis, we studied binding to TG-rich emulsion and biophysical properties of the apoA-I mutants that induce HTG, apoA-I[E110A/E111A] and apoA-I[Δ(61-78)], and compared them to those of WT apoA-I and another apoA-I mutant, apoA-I[Δ(89-99)], that does not induce HTG but causes hypercholesterolemia in mice. We found that the apoA-I[E110A/E111A] and apoA-I[Δ(61-78)] mutations lead to enhanced binding of apoA-I to TG-rich particles, destabilization, and greater exposure of the hydrophobic surface of the protein. The apoA-I[Δ(89-99)] mutant did not show enhanced binding to the emulsion or a more exposed hydrophobic surface. Thus, like apoE4, the apoA-I variants that cause HTG in mice have the altered conformation and stability that facilitate their binding to TG-rich lipoproteins and thereby may lead to the reduced level of lipolysis of these lipoproteins. While many factors may be involved in induction of HTG, we suggest that an increased level of association of destabilized loosely folded apolipoproteins with TG-rich lipoproteins may contribute to some cases of HTG in humans.     

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated.ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion.Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.     

Structure, evolution, and regulation of chicken apolipoprotein A-I

A full-length cDNA clone for the precursor form of chicken liver apolipoprotein A-I (apoA-I) was isolated by antibody screening of a chicken liver cDNA library in the expression vector lambda gt11. The complete nucleotide sequence and predicted amino acid sequence of this clone is presented. The identity of the clone was confirmed by comparison with partial amino acid sequences for chicken apolipoprotein A-I. Chicken preproapolipoprotein A-1 consists of an 18-amino acid prepeptide, a 6-amino acid propeptide, and 240 amino acids of mature protein. The sequence of the protein is homologous to mammalian apoA-I and is highly internally repetitive, consisting largely of 11-amino acid repeats predicted to have an amphipathic alpha-helical structure. The sequence of the propeptide (Arg-Ser-Phe-Trp-Gln-His) differs in two positions from that of mammalian apoA-I. The mRNA for chicken apoA-I is about 1 kilobase in length and is expressed in a variety of tissues including liver, intestine, brain, adrenals, kidneys, heart, and muscle. This quantitative tissue distribution has been determined and is similar to that observed for mammalian apoE and different from that of mammalian apoA-I mRNA. This reinforces the concept that avian apoA-I performs functions analogous to those of mammalian apoE. Moreover, comparisons revealed sequences of chicken apoA-I similar to the region of mammalian apoE responsible for interaction with cellular receptors. Previous studies have demonstrated striking changes in the rates of synthesis of apoA-I in breast muscle during development and in optic nerve after retinal ablation. We now demonstrate that these changes are paralleled by changes in mRNA levels. ApoA-I mRNA levels increase approximately 50-fold in breast muscle between 14 days postconception and hatching and then decrease about 15-fold to adult levels. The levels of apoA-I mRNA increase about 3-fold in optic nerve following retinal ablation. ApoA-I mRNA is also found in the brain in the absence of nerve injury. This may indicate that locally synthesized apoA-I has a routine or housekeeping function in lipid metabolism in the central nervous system.     

Cholesterol efflux to high-density lipoproteins and apolipoprotein A-I phosphatidylcholine complexes is inhibited by ethanol: role of apolipoprotein structure and cooperative interaction of phosphatidylcholine and cholesterol

There is a substantial body of evidence showing that moderate alcohol consumption is associated with a reduced risk of cardiovascular morbidity and mortality. One of the factors thought to contribute to this reduction in risk is an increase in the level of high-density lipoproteins (HDL) correlated with alcohol consumption. However, HDL levels are elevated in heavy drinkers, but their risk of vascular disease is greater compared with that of moderate drinkers. Ethanol at concentrations observed in heavy drinkers and alcoholics may directly act on HDL and apolipoproteins and in turn modify cholesterol efflux. In this paper, we show that ethanol significantly inhibited cholesterol efflux from fibroblasts to HDL and to apolipoprotein A-I (apoA-I) complexed with phosphatidylcholine (PC). Ethanol significantly inhibited binding of PC to apoA-I, inhibited incorporation of cholesterol only when apoA-I contained PC, and did not alter incorporation of cholesterol into HDL. ApoA-I structure was altered by ethanol as monitored by steady-state fluorescence polarization of tryptophan residues. The absence of ethanol effects on incorporation of cholesterol into HDL versus inhibition of cholesterol incorporation into the apoA-I-PC complex suggests that the effects of ethanol on cholesterol efflux mediated by HDL involve interaction with the cell surface and that efflux mediated by the apoA-I-PC complex is a combination of aqueous diffusion and contact with the cell surface. In addition, effects of ethanol on apoA-I suggest that pre-beta-HDL or lipid-free apoA-I may be more perturbed by ethanol than mature HDL, and such effects may be pathophysiological with respect to the process of reverse cholesterol transport in heavy drinkers and alcoholics.