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.
To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.
Objective-The purpose of this study was to understand the interactions of apoA-I with cells expressing ABCA1. Methods and Results-The binding of wild-type (WT) and mutant forms of human apoA-I to mouse J774 macrophages was examined. Analysis of total binding at 37°C of 125 I-WT apoA-I to the cells and specifically to ABCA1, as determined by covalent cross-linking, revealed saturable high affinity binding in both cases. Determination of the level of cell-surface expression of ABCA1 showed that only about 10% of the apoA-I associated with the cell surface was bound directly to ABCA1. Furthermore, when 125 I -apoA-I was cross-linked to ABCA1-upregulated cells and examined by SDS-PAGE, the major (Ϸ90%) band migrated as monomeric apoA-I. In contrast to WT apoA-I, the C-terminal deletion mutants ⌬190 to 243 and ⌬223 to 243 that have reduced lipid affinity, exhibited marked reductions (50 and 70%, respectively) in their abilities to bind to the surface of ABCA1-upregulated cells. However, these C-terminal deletion mutants cross-linked to ABCA1 as effectively as WT apoA-I. Conclusions-This study demonstrates that ABCA1 activity creates 2 types of high affinity apoA-I binding sites at the cell surface. The low capacity site formed by direct apoA-I/ABCA1 interaction functions in a regulatory role, whereas the much higher capacity site generated by apoA-I/lipid interactions functions in the assembly of nascent HDL particles. Key Words: ABCA1 Ⅲ apoA-I Ⅲ phospholipids Ⅲ binding H igh density lipoprotein (HDL) is known to possess pleiotropic biological properties which are able to protect against the development of cardiovascular disease. 1-3 An important aspect of HDL function is its role in reverse cholesterol transport (RCT), a process that mediates the transport of cholesterol from peripheral tissues, including the arterial wall, to the liver for biliary secretion. 1,4,5 Cellular lipid efflux mediated by apolipoprotein (apo) A-I, the major HDL protein constituent, is one of the earliest events in RCT.Recent studies have identified the ATP-binding cassette transporter A1 (ABCA1) as a critical factor in the formation of HDL, and lack of a functional ABCA1 leads to cholesterol accumulation in peripheral tissues and low plasma HDL. 6 The importance of ABCA1 has been demonstrated by the fact that mutations in the ABCA1 gene lead to Tangier disease and familial HDL deficiency. 7 These patients are generally characterized by an accumulation of excess cellular cholesterol, low levels of HDL, and an elevated risk of atherosclerosis. 8 Indeed, a direct correlation between cell surface expression of ABCA1 and apoA-I-mediated cellular lipid efflux has been revealed. 4 Although it is known that the interaction of apoA-I with functional ABCA1 results in the lipidation of apoA-I and formation of HDL, the mechanism of this interaction is not resolved. Prior studies 9 -11 have established that the initial binding of apoA-I to the surface of cells in which ABCA1 is upregulated directs a sequence of events leading to the transfer of ce...
Apolipoprotein (apo) E4 has been identified as a major risk factor for Alzheimer's disease. Recently, apoE4 was found to undergo proteolytic cleavage in Alzheimer's disease brains, resulting in neurotoxic C-terminal-truncated fragments. In this study, we examined the effect of progressive truncation of the C-terminal domain in apoE4 on its lipid-free structure and lipid binding properties. Circular dichroism measurements demonstrated that deletion of residues 273-299 or 261-299 significantly decreased the number of helical residues, suggesting that the C-terminal residues 261-299 have alpha-helical structure. Although the progressive deletions in the C-terminal domain appear to somewhat increase thermal stability, apoE4 (delta273-299) and apoE4 (delta261-299) showed stability similar to that of the apoE4 22-kDa fragment (residues 1-191) when denatured with guanidine-HCl, indicating that residues 192-272 have a negligible effect on the stability of the C-terminal-truncated apoE4. Comparison of Trp-264 fluorescence in single Trp mutants of full-length and C-terminal-truncated apoE4 (delta273-299) indicated that the C-terminal domain structure in the latter is both less organized and cooperative. In addition, comparison of the binding of the C-terminal-truncated mutants to a hydrophobic fluorescent dye and to lipid emulsions revealed that residues 261-272 create a hydrophobic site which is critical for lipid binding. These results suggest that removal of a hydrophobic C-terminal alpha-helical segment (residues 273-299) to create C-terminal-truncated apoE4 forms found in brain leads to less organized C-terminal structure while still retaining a second alpha-helical lipid-binding region (residues 261-272) that is available for interaction with cell membranes and other proteins such as amyloid beta peptide.
This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.
The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux (ϳ90%), indicating that the C-terminal amphipathic ␣-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux (ϳ30%) and increased the K m about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (⌬123-166) had no effect on either K m or V max . These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic ␣-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.
Objective-To compare the abilities of human wild-type apoA-I (WT apoA-I) and human apoA-I Milano (apoA-I M ) to promote macrophage reverse cholesterol transport (RCT) in apoA-I-null mice infected with adeno-associated virus (AAV) expressing either WT apoA-I or apoA-I M . Methods and Results-WT apoA-I-or apoA-I M -expressing mice were intraperitoneally injected with [H 3 ]cholesterollabeled J774 mouse macrophages. After 48 hours, no significant difference was detected in the amount of cholesterol removed from the macrophages and deposited in the feces via the RCT pathway between the WT apoA-I and apoA-I M groups. Analysis of the individual components of the RCT pathway demonstrated that the apoA-I M -expressing mice promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux as efficiently as WT apoA-I but that apoA-I M had a reduced ability to promote cholesterol esterification via lecithin cholesterol-acyltransferase (LCAT). This resulted in reduced cholesteryl ester (CE) and increased free cholesterol (FC) levels in the plasma of mice expressing apoA-I M compared to WT apoA-I. These differences did not affect the rate of delivery of labeled cholesterol to the liver via SR-BI-mediated selective uptake or its subsequent excretion in the feces. Key Words: apoA-I Milano Ⅲ reverse cholesterol transport Ⅲ high-density lipoprotein Ⅲ macrophage Ⅲ apolipoprotein A polipoprotein A-I (apoA-I) is the major protein component of HDL. 1,2 Plasma apoA-I and HDL cholesterol levels are inversely associated with the risk of cardiovascular disease. 3 The antiatherogenic properties of apoA-I are thought to arise, at least in part, from its central role in reverse cholesterol transport (RCT), the pathway by which excess cholesterol is effluxed from peripheral tissues and transported to the liver, where it is subsequently excreted from the body. [3][4][5] The first natural mutant of apoA-I identified was the apoA-I Milano (apo A-I M ) mutation (R173C). 6 The apoA-I M mutation is located in the N-terminal helix bundle domain of the protein. 7,8 Naturally occurring variants of apoA-I between residues 121 and 186 are often associated with low apoA-I levels. 9 Individuals heterozygous for the apoA-I M mutation have very low plasma apoA-I and HDL cholesterol levels as well as moderately elevated triglycerides. 10 Despite a lipid profile that is usually associated with a high risk of premature cardiovascular disease, apoA-I M carriers display no increase in cardiovascular disease or events. 10 -12 This has led to speculation that apoA-I M is a gain-of-function mutation that has enhanced cardio-protective effects, [13][14][15][16][17][18][19] whereas others believe that wild-type (WT) apoA-I and apoA-I M are functionally equivalent. 20,21 A clinical trial of repeated intravenous infusions of apoA-I M -phospholipid complexes demonstrated regression of existing atheromas after 5 weekly treatments, 22,23 whereas other clinical trials focused on the ability of WT apoA-I/phosphatidylcholine disks to increase endotheli...
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