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.     

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.     

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.     

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins.

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of prebeta HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between alpha and prebeta. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.     

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.     

Modifying Apolipoprotein A-I by Malondialdehyde,but Not by an Array of Other Reactive Carbonyls,Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.     

Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.     

Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.     

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of preβ HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between α and preβ. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.     

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.     

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1–189 and 190–243) and mouse apoA-I (residues 1–186 and 187–240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1

It is widely accepted that functional ATP-binding cassette transporter A1 (ABCA1) is critical for the formation of nascent high density lipoprotein particles. However, the cholesterol pool(s) and the cellular signaling processes utilized by the ABCA1-mediated pathway remain unclear. Sphingomyelin maintains a preferential interaction with cholesterol in membranes, and its catabolites, especially ceramide, are potent signaling molecules that could play a role in ABCA1 regulation or function. To study the potential role of ceramide in this process, we treated a variety of cell lines with 20 microM C2-ceramide and examined apolipoprotein-mediated cholesterol efflux to lipid-free apoA-I. We found that cell lines expressing ABCA1 displayed 2-3-fold increases in cholesterol efflux to apoA-I. Cell lines not expressing ABCA1 were unaffected by ceramide. We further characterized the cholesterol efflux effect in Chinese hamster ovary cells. Ceramide treatment did not cause significant cytotoxicity or apoptosis and did not affect cholesterol efflux to non-apolipoprotein acceptors. Raising endogenous ceramide levels increased cholesterol efflux to apoA-I. Using a cell surface biotinylation method, we found that the total cellular ABCA1 and that at the plasma membrane were increased with ceramide treatment. Also ceramide enhanced the binding of fluorescently labeled apoA-I to Chinese hamster ovary cells. These data suggest that ceramide may increase the plasma membrane content of ABCA1, leading to increased apoA-I binding and cholesterol efflux.     

ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux

Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (Kd of 7.0 +/- 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.     

ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.     

Probucol inactivates ABCA1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-I-mediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.     

ABCA1 and ABCG1 or ABCG4 act sequentially to remove cellular cholesterol and generate cholesterol-rich HDL

Recent developments in lipid metabolism have shown the importance of ATP binding cassette transporters (ABCs) in controlling cellular and total body lipid homeostasis. ABCA1 mediates the transport of cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I), whereas ABCG1 and ABCG4 mediate the transport of cholesterol from cells to lipidated lipoproteins. ABCA1, ABCG1, and ABCG4 are all expressed in cholesterol-loaded macrophages, and macrophages from ABCA1 and ABCG1 knockout mice accumulate cholesteryl esters. Here, we show that the lipidated particles generated by incubating cells overexpressing ABCA1 with apoA-I are efficient acceptors for cholesterol released from cells overexpressing either ABCG1 or ABCG4. The cholesterol released to the particles was derived from a cholesterol oxidase-accessible plasma membrane pool in both ABCG1 and ABCG4 cells, which is the same pool of cholesterol shown previously to be removed by high density lipoproteins. ABCA1 cells incubated with apoA-I generated two major populations of cholesterol- and phospholipid-rich lipoprotein particles that were converted by ABCG1 or ABCG4 cells to one major particle population that was highly enriched in cholesterol. These results suggest that ABCG1 and ABCG4 act in concert with ABCA1 to maximize the removal of excess cholesterol from cells and to generate cholesterol-rich lipoprotein particles.     

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.