An amphipathic helical region of the N-terminal barrel of phospholipid transfer protein is critical for ABCA1-dependent cholesterol efflux

Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.     

ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation

ABCA1 exports cholesterol and phospholipids from cells by a multistep pathway that involves forming cell surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Here we used a mutational analysis approach to evaluate the relationship between these events. We prepared seven naturally occurring mutants and one artificial missense mutant of ABCA1 with varying degrees of impaired function, expressed them to similar levels as wild-type ABCA1 on the cell surface of BHK cells, and measured ABCA1-dependent lipid export, apolipoprotein A-I (apoA-I) binding, and signaling activities. Linear regression analyses showed that cholesterol and phospholipid efflux and cellular apoA-I binding correlated significantly with the ability of ABCA1 to form cell surface lipid domains. Lipid export and cellular apoA-I binding activities and formation of lipid domains also correlated with the amount of apoA-I that could be cross-linked to ABCA1. Moreover, each of these lipid export and apoA-I binding activities correlated with apoA-I-induced Janus kinase 2 (JAK2) activation. Thus, these missense mutations in ABCA1 impair lipid export, apoA-I binding, and apoA-I-stimulated JAK2 activities to similar extents, indicating that these processes are highly interactive components of a pathway that functions to export lipids from cells.     

Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol

ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.     

ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions

ATP binding cassette transporter A1 (ABCA1) mediates the transport of phospholipids and cholesterol from cells to lipid-poor HDL apolipoproteins. Cholesterol loading of cells induces ABCA1, implicating cholesterol as its major physiologic substrate. It is believed, however, that ABCA1 is primarily a phospholipid transporter and that cholesterol efflux occurs by diffusion to ABCA1-generated phospholipid-rich apolipoproteins. Here we show that overexpression of ABCA1 in baby hamster kidney cells in the absence of apolipoproteins redistributed membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. The cholesterol removed by apolipoprotein A-I (apoA-I), but not by HDL phospholipids, was derived exclusively from these domains. ABCA1 overexpression also increased cholesterol esterification, which was prevented by addition of apoA-I, suggesting that some of the cell-surface cholesterol not removed by apolipoproteins is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. ABCA1 expression was essential for cholesterol efflux even when apolipoproteins had already acquired phospholipids during prior exposure to ABCA1-expressing cells.These studies show that ABCA1 redistributes cholesterol to cell-surface domains, where it becomes accessible for removal by apolipoproteins, consistent with a direct role of ABCA1 in cholesterol transport.     

Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A1 and enhances cholesterol efflux from cells

Phospholipid lipid transfer protein (PLTP) is ubiquitously expressed in animal tissues and plays multiple roles in lipoprotein metabolism, but the function of peripheral PLTP is still poorly understood. Here we show that one of its possible functions is to transport cholesterol and phospholipids from cells to lipoprotein particles by a process involving PLTP interactions with cellular ATP-binding cassette transporter A1 (ABCA1). When ABCA1 was induced in murine macrophages or ABCA1-transfected baby hamster kidney cells, PLTP gained the ability to promote cholesterol and phospholipid efflux from cells. Although PLTP alone had lipid efflux activity, its maximum activity was observed in the presence of high density lipoprotein particles. Pulsechase studies showed that the interaction of PLTP with ABCA1-expressing cells played a role in promoting lipid efflux. Overexpression of ABCA1 dramatically increased binding of both PLTP and apoA-I to common sites on the cell surface. Both PLTP and apoA-I were covalently cross-linked to ABCA1, each protein blocked cross-linking of the other, and both PLTP and apoA-I stabilized ABCA1 protein. These results are consistent with PLTP and apoA-I binding to ABCA1 at the same or closely related sites. Thus, PLTP mimics apolipoproteins in removing cellular lipids by the ABCA1 pathway, except that PLTP acts more as an intermediary in the transfer of cellular lipids to lipoprotein particles.     

ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein

ABCA1, an ATP-binding cassette transporter mutated in Tangier disease, promotes cellular phospholipid and cholesterol efflux by loading free apoA-I with these lipids. This process involves binding of apoA-I to the cell surface and phospholipid translocation by ABCA1. The goals of this study were to examine the relationship between ABCA1-mediated lipid efflux and apolipoprotein binding and to determine whether phospholipid and cholesterol efflux are coupled. Inhibition of lipid efflux by glybenclamide treatment or by mutation of the ATP-binding cassette of ABCA1 showed a close correlation between lipid efflux, the binding of apoA-I to cells, and cross-linking of apoA-I to ABCA1. The data suggest that a functionally important apoA-I binding site exists on ABCA1 and that the binding site could also involve lipids. After using cyclodextrin preincubation to deplete cellular cholesterol, ABCA1-mediated cholesterol efflux was abolished but phospholipid efflux and the binding of apoA-I were unaffected. The conditioned media from cyclodextrin-pretreated, ABCA1-expressing cells readily promoted cholesterol efflux when added to fresh cells not expressing ABCA1, indicating that cholesterol efflux can be dissociated from phospholipid efflux. Further, using a photoactivatable cholesterol analog, we showed that ABCA1 did not bind cholesterol directly, even though several other cholesterol-binding proteins specifically bound the cholesterol analog. The data suggest that the binding of apoA-I to ABCA1 leads to the formation of phospholipid-apoA-I complexes, which subsequently promote cholesterol efflux in an autocrine or paracrine fashion.     

Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: an important determinant for their distinctive role in lipid metabolism

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.     

Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tangier disease and ABCA1

Tangier disease is an autosomal recessive genetic disorder characterized by a severe high-density lipoprotein (HDL) deficiency, sterol deposition in tissue macrophages, and prevalent atherosclerosis. Mutations in the ATP binding cassette transporter ABCA1 cause Tangier disease and other familial HDL deficiencies. ABCA1 controls a cellular pathway that secretes cholesterol and phospholipids to lipid-poor apolipoproteins. This implies that an inability of newly synthesized apolipoproteins to acquire cellular lipids by the ABCA1 pathway leads to their rapid degradation and an over-accumulation of cholesterol in macrophages. Thus, ABCA1 plays a critical role in modulating flux of tissue cholesterol and phospholipids into the reverse cholesterol transport pathway, making it an important therapeutic target for clearing excess cholesterol from macrophages and preventing atherosclerosis.     

Helical apolipoproteins of high-density lipoprotein enhance phagocytosis by stabilizing ATP-binding cassette transporter A7

We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.     

Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1

ABCA1 mediates the transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Apolipoprotein A-I (apoA-I) interactions with ABCA1-expressing cells elicit several responses, including removing cellular lipids, stabilizing ABCA1 protein, and activating Janus kinase 2 (JAK2). Here, we used synthetic apolipoprotein-mimetic peptides to characterize the relationship between these responses. Peptides containing one amphipathic helix of L- or D-amino acids (2F, D-2F, or 4F) and a peptide containing two helices (37pA) all promoted ABCA1-dependent cholesterol efflux, competed for apoA-I binding to ABCA1-expressing cells, blocked covalent cross-linking of apoA-I to ABCA1, and inhibited ABCA1 degradation. 37pA was cross-linked to ABCA1, confirming the direct binding of amphipathic helices to ABCA1. 2F, 4F, 37pA, and D-37pA all stimulated JAK2 autophosphorylation. Inhibition of JAK2 greatly reduced peptide-mediated cholesterol efflux, peptide binding to ABCA1-expressing cells, and peptide cross-linking to ABCA1, indicating that these processes require an active JAK2. In contrast, apoA-I and peptides stabilized ABCA1 protein even in the absence of an active JAK2, implying that this process is independent of JAK2 and lipid efflux-promoting binding of amphipathic helices to ABCA1. These findings show that amphipathic helices coordinate the activity of ABCA1 by several distinct mechanisms that are likely to involve different cell surface binding sites.     

Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux

The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37 degrees C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.