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Papers by Maxime Denis

Research paper thumbnail of Cellular Physiology of Cholesterol Efflux in Vascular Endothelial Cells

Circulation, Nov 2, 2004

Background-Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are t... more Background-Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis. Methods and Results-We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1-mediated endothelial cholesterol efflux. Conclusions-Endothelial cells may use nontraditional pathways for cholesterol efflux.

Research paper thumbnail of Régulation moléculaire et cellulaire de l'efflux de cholestérol par le transporteur ATP-binding cassette A1(ABCA1)

des niveaux de lipoprotéines de haute densité (H DL) anormalement bas, un facteur de risque pour ... more des niveaux de lipoprotéines de haute densité (H DL) anormalement bas, un facteur de risque pour le développement de maladies cardiovasculaires. In vivo, les HDL ont un effet athéroprotecteur important puisqu'elles effectuent le transport à rebours du cholestérol des tissus périphériques vers le foie. Or, la maladie de Tangier est causée par des mutations dans le gène du transporteur « ATP-binding cassette Al (ABCA1). Le modèle actuel veut que ce transporteur promeuve la lipidation de l'apolipoprotéine A-l (apoA-l), la composante protéique majeure des HDL, pour former des particules HDL naissantes discoïdales ayant une migration électrophorétique en position pré-f3. Un défaut dans la lipidation de l'apoA-I par l'ABCAl abolit la biogénèse des HDL. Aussi, nous avons voulu étudier la régulation transcriptionnelle de l'ABCAl, son interaction avec son ligand (l'apoA-l), les particules qui en résultent, et la conformation structurale requise du transporteur pour effectuer sa fonction. D'abord, en utilisant un inhibiteur de facteurs nucléaires spécifiques, il a été établi qu'une charge en cholestérol induit la transcription d'ABCAl via la génération d'hydroxystérols. Ensuite, nous avons utilisé ce système inductible afin de déterminer la nature du contact entre l'apoA-l et l'ABCAl. Une étude de radioliaison avec prétraitement des membranes cellulaires aux phospholipases n'affecte pas la liaison, excluant une interaction indirecte médiée par des domaines lipidiques. Contrairement au modèle proposé plus haut, nous avons établi par gel bi-dimensionnel que la lipidation de l'apoA-l par l'ABCAl génère des HDL naissantes migrant plutôt en position alpha. Finalement, par pontage moléculaire nous avons montré que la liaison de l'apoA-l requière l'organisation de l'ABCAl en une structure oligomérique. Cette oligomérisation résulte en la formation de particules alpha composées de plusieurs molécules d'apoA-I. Ces observations réunies démontrent que 1) l'apoA-l s'associe à l'ABCAl oligomérique dans une interaction protéine!protéine directe; 2) cette interaction résulte en la formation de HDL naissantes contenant plusieurs molécules d'apoA-I phospholipidées migrant en position alpha. En conclusion, les résultats présentés dans ce travail bouleversent complètement les modèles préalablement établis et proposent de nouveaux concepts et avenues de recherche sur l'homéostasie du cholestérol par les HDL.

Research paper thumbnail of Abstract 5567: The Ldlr -/- Mouse as a Novel Model of Aortic Calcification: Application of Micro-Computerized Tomography

Research paper thumbnail of Reducing Vascular Calcification by Anti-IL-1β Monoclonal Antibody in a Mouse Model of Familial Hypercholesterolemia

Angiology, 2015

Background: Given the link between cholesterol and activation of inflammation via interleukin 1β ... more Background: Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr−/−) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. Methods: A mouse monoclonal antibody (mAb) against IL-1β or placebo was administered subcutaneously in Ldlr−/− and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 ...

Research paper thumbnail of High density lipoproteins in health and in disease

Journal of investigative medicine : the official publication of the American Federation for Clinical Research, 1999

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Research paper thumbnail of Annexin A2 Is a Natural Extrahepatic Inhibitor of the PCSK9-Induced LDL Receptor Degradation

PLoS ONE, 2012

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density ... more Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2 2/2 mice revealed: i) a ,1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ,2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2 2/2 tissues revealed that the LDLR was decreased by ,50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9;LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

Research paper thumbnail of Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

Journal of Lipid Research, 2005

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic propert... more It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣-nascent apolipoprotein A-I-containing particles (␣-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both ␣-LpA-I and pre ␤ 1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of ␣-LpA-I but not pre ␤ 1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both pre ␤ 1-LpA-I and ␣-LpA-I; by contrast, CaCo-2 cells secreted only ␣-LpA-I. To determine whether ␣-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated ␣-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V max (8.4 vs. 8.2 nmol cholesteryl ester/h/ g LCAT, respectively), the K m value was increased 2-fold for ␣-LpA-I compared with r(HDL) (1.2 vs. 0.7 M apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of ␣-LpA-I but not pre ␤ 1-LpA-I; and 2) ␣-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and-independent pathways.-Krimbou, L.

Research paper thumbnail of Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

Journal of Lipid Research, 2007

Research paper thumbnail of Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B

Journal of Lipid Research, 2005

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL.... more We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3. Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.-Lee, C.

Research paper thumbnail of Molecular interactions between apoE and ABCA1

Journal of Lipid Research, 2004

Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced ... more Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipidfree apoE3 inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC 50 ‫؍‬ 2.5 ؎ 0.4 g/ml vs. 12.3 ؎ 1.3 g/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/ cholesterol/phospholipid complexes that exhibited pre ␤electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high densitysized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.

Research paper thumbnail of cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts

Journal of Lipid Research, 2002

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step... more ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in Ͻ 10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dosedependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.-Haidar, B., M. Denis, L.

Research paper thumbnail of Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

Journal of Lipid Research, 2005

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apo... more It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates pre ␤ 1-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated pre ␤ 1-LpA-I-like particles inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than HDL 3 (IC 50 ‫؍‬ 2.20 ؎ 0.35 vs. 37.60 ؎ 4.78 g/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased (‫ف‬ 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native pre ␤ 1-LpA-I and pre ␤ 1-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither pre ␤ 1-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from ␣-HDL to pre ␤-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma pre ␤ 1-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

Research paper thumbnail of Apolipoprotein A-I Activates Cellular cAMP Signaling through the ABCA1 Transporter

Journal of Biological Chemistry, 2003

Research paper thumbnail of Molecular and Cellular Physiology of Apolipoprotein A-I Lipidation by the ATP-binding Cassette Transporter A1 (ABCA1)

Journal of Biological Chemistry, 2003

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts in... more The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of 125 I-apoA-I to ABCA1 at 37°C were determined: K d ‫؍‬ 0.65 g/ml, B max ‫؍‬ 0.10 ng/g cell protein. Lipid-free apoA-I inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than pre-␤ 1-LpA-I, reconstituted HDL particles r(LpA-I), or HDL 3 (IC 50 ‫؍‬ 0.35 ؎ 1.14, apoA-I; 1.69 ؎ 1.07, pre-␤ 1-LpA-I; 17.91 ؎ 1.39, r(LpA-I); and 48.15 ؎ 1.72 g/ ml, HDL 3). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither 125 I-apoA-I binding nor 125 I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of 125 I-apoA-I from normal cells at 37°C was rapid (t1 ⁄2 ‫؍‬ 1.4 ؎ 0.66 h; n ‫؍‬ 3) but almost completely inhibited at either 15 or 4°C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates ␣-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis. Apolipoprotein (apo) 1 A-I binding to the extracellular domain of ABCA1 results in the activation of apoA-I lipidation, a key

Research paper thumbnail of Cellular Physiology of Cholesterol Efflux in Vascular Endothelial Cells

Circulation, 2004

Background— Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are ... more Background— Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis. Methods and Results— We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1–mediated endothelial cholesterol efflux. Conclusions— Endothelial cells may use nontraditional pathways for cholesterol efflux.

Research paper thumbnail of Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice

Circulation, 2012

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of i... more Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice...

Research paper thumbnail of Ht31, a PKA anchoring inhibitor, induces robust cholesterol efflux and reverses macrophage foam cell formation through ABCA1

Journal of Biological …, 2010

... Loretta Ma2, Fumin Dong1, Maxime Denis1, Ying Feng1, Ming-Dong Wang3 and Xiaohui Zha1, 2 ... ... more ... Loretta Ma2, Fumin Dong1, Maxime Denis1, Ying Feng1, Ming-Dong Wang3 and Xiaohui Zha1, 2 ... 21. Sethi, AA, Stonik, JA, Thomas, F., Demosky, SJ, Amar, M., Neufeld, E., Brewer, HB, Davidson, WS, D'Souza, W., Sviridov, D., and Remaley, AT (2008) J. Biol. Chem. ...

Research paper thumbnail of Characterization of Oligomeric Human ATP Binding Cassette Transporter A1

Journal of Biological Chemistry, 2004

The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nasce... more The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-Icontaining particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I with diameters between 9.5 and 20 nm. Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. These results demonstrate that: 1) ABCA1 exists as an oligomeric complex; and 2) ABCA1 oligomerization was independent of apoA-I binding and lipid molecules. The findings that the majority of ABCA1 exists as a tetramer that binds apoA-I, together with the observation that LpA-I contains at least four molecules of apoA-I per particle, support the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit required for the biogenesis of high density lipoprotein particles.

Research paper thumbnail of Abstract 1385: The ATP-binding Cassette A1 (ABCA1)-mediated Apolipoprotein A-I Lipidation Occurs at the Plasma Membrane and not in the Endocytic Compartments

Circulation, Oct 16, 2007

<jats:p> The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of ... more <jats:p> The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of HDL through the lipidation of lipid-poor apolipoprotein A-I (apoA-I). ApoA-I has been suggested to internalize with ABCA1, presumably to acquire lipids from the endosomal compartments, an important hub for cholesterol trafficking. The aim of our work is to determine how apoA-I get endocytosed and whether this internalization contributes to the biogenesis of HDL. When examined by confocal microscopy, we found that Cy3.5-apoA-I endocytosed rather slowly (t <jats:sub>1/2</jats:sub> = 15 min, in baby hamster kidney cells expressing ABCA1) and poorly co-localized with transferrin, consistent with a pathway independent of clathrin-coated pits. ApoA-I was instead found perfectly co-localized with FITC-dextran, a bulk phase uptake marker and, at later time points, with LysoTracker. This strongly indicates that majority of internalized apoA-I was delivered to the lysosomes. ABCA1 was not found to co-localize with endocytosed apoA-I. Similar observations were obtained in mouse macrophages and normal human fibroblasts. Next, in order to determine the functional significance of endocytosis to cholesterol efflux, we used sucrose or latrunculin A to inhibit apoA-I endocytosis. We show that apoA-I, transferrin and dextran uptake were abolished, but cholesterol efflux was not decreased. To specifically determine whether internalized apoA-I contributes to the biogenesis of HDL, we developed a method to strip off apoA-I from the cell surface. Our results show that internalized apoA-I do not significantly contribute to the biogenesis of HDL (&lt; 20%). Together, our results suggest that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs. Internalized apoA-I is mostly targeted for lysosomal degradation and therefore does not significantly contribute to the biogenesis of HDL. </jats:p>

Research paper thumbnail of Cholesterol Is Required for Efficient Endoplasmic Reticulum-to-Golgi Transport of Secretory Membrane Proteins

Molecular Biology of the Cell, 2006

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellul... more Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3–6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss...

Research paper thumbnail of Cellular Physiology of Cholesterol Efflux in Vascular Endothelial Cells

Circulation, Nov 2, 2004

Background-Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are t... more Background-Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis. Methods and Results-We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1-mediated endothelial cholesterol efflux. Conclusions-Endothelial cells may use nontraditional pathways for cholesterol efflux.

Research paper thumbnail of Régulation moléculaire et cellulaire de l'efflux de cholestérol par le transporteur ATP-binding cassette A1(ABCA1)

des niveaux de lipoprotéines de haute densité (H DL) anormalement bas, un facteur de risque pour ... more des niveaux de lipoprotéines de haute densité (H DL) anormalement bas, un facteur de risque pour le développement de maladies cardiovasculaires. In vivo, les HDL ont un effet athéroprotecteur important puisqu'elles effectuent le transport à rebours du cholestérol des tissus périphériques vers le foie. Or, la maladie de Tangier est causée par des mutations dans le gène du transporteur « ATP-binding cassette Al (ABCA1). Le modèle actuel veut que ce transporteur promeuve la lipidation de l'apolipoprotéine A-l (apoA-l), la composante protéique majeure des HDL, pour former des particules HDL naissantes discoïdales ayant une migration électrophorétique en position pré-f3. Un défaut dans la lipidation de l'apoA-I par l'ABCAl abolit la biogénèse des HDL. Aussi, nous avons voulu étudier la régulation transcriptionnelle de l'ABCAl, son interaction avec son ligand (l'apoA-l), les particules qui en résultent, et la conformation structurale requise du transporteur pour effectuer sa fonction. D'abord, en utilisant un inhibiteur de facteurs nucléaires spécifiques, il a été établi qu'une charge en cholestérol induit la transcription d'ABCAl via la génération d'hydroxystérols. Ensuite, nous avons utilisé ce système inductible afin de déterminer la nature du contact entre l'apoA-l et l'ABCAl. Une étude de radioliaison avec prétraitement des membranes cellulaires aux phospholipases n'affecte pas la liaison, excluant une interaction indirecte médiée par des domaines lipidiques. Contrairement au modèle proposé plus haut, nous avons établi par gel bi-dimensionnel que la lipidation de l'apoA-l par l'ABCAl génère des HDL naissantes migrant plutôt en position alpha. Finalement, par pontage moléculaire nous avons montré que la liaison de l'apoA-l requière l'organisation de l'ABCAl en une structure oligomérique. Cette oligomérisation résulte en la formation de particules alpha composées de plusieurs molécules d'apoA-I. Ces observations réunies démontrent que 1) l'apoA-l s'associe à l'ABCAl oligomérique dans une interaction protéine!protéine directe; 2) cette interaction résulte en la formation de HDL naissantes contenant plusieurs molécules d'apoA-I phospholipidées migrant en position alpha. En conclusion, les résultats présentés dans ce travail bouleversent complètement les modèles préalablement établis et proposent de nouveaux concepts et avenues de recherche sur l'homéostasie du cholestérol par les HDL.

Research paper thumbnail of Abstract 5567: The Ldlr -/- Mouse as a Novel Model of Aortic Calcification: Application of Micro-Computerized Tomography

Research paper thumbnail of Reducing Vascular Calcification by Anti-IL-1β Monoclonal Antibody in a Mouse Model of Familial Hypercholesterolemia

Angiology, 2015

Background: Given the link between cholesterol and activation of inflammation via interleukin 1β ... more Background: Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr−/−) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. Methods: A mouse monoclonal antibody (mAb) against IL-1β or placebo was administered subcutaneously in Ldlr−/− and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 ...

Research paper thumbnail of High density lipoproteins in health and in disease

Journal of investigative medicine : the official publication of the American Federation for Clinical Research, 1999

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Research paper thumbnail of Annexin A2 Is a Natural Extrahepatic Inhibitor of the PCSK9-Induced LDL Receptor Degradation

PLoS ONE, 2012

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density ... more Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2 2/2 mice revealed: i) a ,1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ,2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2 2/2 tissues revealed that the LDLR was decreased by ,50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9;LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

Research paper thumbnail of Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

Journal of Lipid Research, 2005

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic propert... more It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣-nascent apolipoprotein A-I-containing particles (␣-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both ␣-LpA-I and pre ␤ 1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of ␣-LpA-I but not pre ␤ 1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both pre ␤ 1-LpA-I and ␣-LpA-I; by contrast, CaCo-2 cells secreted only ␣-LpA-I. To determine whether ␣-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated ␣-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V max (8.4 vs. 8.2 nmol cholesteryl ester/h/ g LCAT, respectively), the K m value was increased 2-fold for ␣-LpA-I compared with r(HDL) (1.2 vs. 0.7 M apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of ␣-LpA-I but not pre ␤ 1-LpA-I; and 2) ␣-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and-independent pathways.-Krimbou, L.

Research paper thumbnail of Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

Journal of Lipid Research, 2007

Research paper thumbnail of Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B

Journal of Lipid Research, 2005

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL.... more We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3. Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.-Lee, C.

Research paper thumbnail of Molecular interactions between apoE and ABCA1

Journal of Lipid Research, 2004

Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced ... more Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipidfree apoE3 inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC 50 ‫؍‬ 2.5 ؎ 0.4 g/ml vs. 12.3 ؎ 1.3 g/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/ cholesterol/phospholipid complexes that exhibited pre ␤electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high densitysized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.

Research paper thumbnail of cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts

Journal of Lipid Research, 2002

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step... more ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in Ͻ 10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dosedependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.-Haidar, B., M. Denis, L.

Research paper thumbnail of Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

Journal of Lipid Research, 2005

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apo... more It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates pre ␤ 1-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated pre ␤ 1-LpA-I-like particles inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than HDL 3 (IC 50 ‫؍‬ 2.20 ؎ 0.35 vs. 37.60 ؎ 4.78 g/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased (‫ف‬ 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native pre ␤ 1-LpA-I and pre ␤ 1-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither pre ␤ 1-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from ␣-HDL to pre ␤-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma pre ␤ 1-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

Research paper thumbnail of Apolipoprotein A-I Activates Cellular cAMP Signaling through the ABCA1 Transporter

Journal of Biological Chemistry, 2003

Research paper thumbnail of Molecular and Cellular Physiology of Apolipoprotein A-I Lipidation by the ATP-binding Cassette Transporter A1 (ABCA1)

Journal of Biological Chemistry, 2003

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts in... more The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of 125 I-apoA-I to ABCA1 at 37°C were determined: K d ‫؍‬ 0.65 g/ml, B max ‫؍‬ 0.10 ng/g cell protein. Lipid-free apoA-I inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than pre-␤ 1-LpA-I, reconstituted HDL particles r(LpA-I), or HDL 3 (IC 50 ‫؍‬ 0.35 ؎ 1.14, apoA-I; 1.69 ؎ 1.07, pre-␤ 1-LpA-I; 17.91 ؎ 1.39, r(LpA-I); and 48.15 ؎ 1.72 g/ ml, HDL 3). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither 125 I-apoA-I binding nor 125 I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of 125 I-apoA-I from normal cells at 37°C was rapid (t1 ⁄2 ‫؍‬ 1.4 ؎ 0.66 h; n ‫؍‬ 3) but almost completely inhibited at either 15 or 4°C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates ␣-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis. Apolipoprotein (apo) 1 A-I binding to the extracellular domain of ABCA1 results in the activation of apoA-I lipidation, a key

Research paper thumbnail of Cellular Physiology of Cholesterol Efflux in Vascular Endothelial Cells

Circulation, 2004

Background— Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are ... more Background— Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis. Methods and Results— We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1–mediated endothelial cholesterol efflux. Conclusions— Endothelial cells may use nontraditional pathways for cholesterol efflux.

Research paper thumbnail of Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice

Circulation, 2012

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of i... more Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice...

Research paper thumbnail of Ht31, a PKA anchoring inhibitor, induces robust cholesterol efflux and reverses macrophage foam cell formation through ABCA1

Journal of Biological …, 2010

... Loretta Ma2, Fumin Dong1, Maxime Denis1, Ying Feng1, Ming-Dong Wang3 and Xiaohui Zha1, 2 ... ... more ... Loretta Ma2, Fumin Dong1, Maxime Denis1, Ying Feng1, Ming-Dong Wang3 and Xiaohui Zha1, 2 ... 21. Sethi, AA, Stonik, JA, Thomas, F., Demosky, SJ, Amar, M., Neufeld, E., Brewer, HB, Davidson, WS, D'Souza, W., Sviridov, D., and Remaley, AT (2008) J. Biol. Chem. ...

Research paper thumbnail of Characterization of Oligomeric Human ATP Binding Cassette Transporter A1

Journal of Biological Chemistry, 2004

The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nasce... more The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-Icontaining particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I with diameters between 9.5 and 20 nm. Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. These results demonstrate that: 1) ABCA1 exists as an oligomeric complex; and 2) ABCA1 oligomerization was independent of apoA-I binding and lipid molecules. The findings that the majority of ABCA1 exists as a tetramer that binds apoA-I, together with the observation that LpA-I contains at least four molecules of apoA-I per particle, support the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit required for the biogenesis of high density lipoprotein particles.

Research paper thumbnail of Abstract 1385: The ATP-binding Cassette A1 (ABCA1)-mediated Apolipoprotein A-I Lipidation Occurs at the Plasma Membrane and not in the Endocytic Compartments

Circulation, Oct 16, 2007

<jats:p> The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of ... more <jats:p> The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of HDL through the lipidation of lipid-poor apolipoprotein A-I (apoA-I). ApoA-I has been suggested to internalize with ABCA1, presumably to acquire lipids from the endosomal compartments, an important hub for cholesterol trafficking. The aim of our work is to determine how apoA-I get endocytosed and whether this internalization contributes to the biogenesis of HDL. When examined by confocal microscopy, we found that Cy3.5-apoA-I endocytosed rather slowly (t <jats:sub>1/2</jats:sub> = 15 min, in baby hamster kidney cells expressing ABCA1) and poorly co-localized with transferrin, consistent with a pathway independent of clathrin-coated pits. ApoA-I was instead found perfectly co-localized with FITC-dextran, a bulk phase uptake marker and, at later time points, with LysoTracker. This strongly indicates that majority of internalized apoA-I was delivered to the lysosomes. ABCA1 was not found to co-localize with endocytosed apoA-I. Similar observations were obtained in mouse macrophages and normal human fibroblasts. Next, in order to determine the functional significance of endocytosis to cholesterol efflux, we used sucrose or latrunculin A to inhibit apoA-I endocytosis. We show that apoA-I, transferrin and dextran uptake were abolished, but cholesterol efflux was not decreased. To specifically determine whether internalized apoA-I contributes to the biogenesis of HDL, we developed a method to strip off apoA-I from the cell surface. Our results show that internalized apoA-I do not significantly contribute to the biogenesis of HDL (&lt; 20%). Together, our results suggest that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs. Internalized apoA-I is mostly targeted for lysosomal degradation and therefore does not significantly contribute to the biogenesis of HDL. </jats:p>

Research paper thumbnail of Cholesterol Is Required for Efficient Endoplasmic Reticulum-to-Golgi Transport of Secretory Membrane Proteins

Molecular Biology of the Cell, 2006

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellul... more Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3–6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss...