ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.     

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

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

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.     

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.     

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E

Gallbladder epithelial cells (GBEC) are exposed to high and fluctuating concentrations of biliary cholesterol on their apical (AP) surface. GBEC absorb and efflux cholesterol, but the mechanisms of cholesterol uptake, intracellular trafficking, and efflux in these cells are not known. We previously reported that ATP binding cassette (ABC)A1 mediates basolateral (BL) cholesterol efflux in cultured polarized GBEC. In addition, the nuclear hormone receptors liver X receptor (LXR)alpha and retinoid X receptor (RXR) mediate both AP and BL cholesterol efflux. An interesting finding from our previous study was that apolipoprotein (apo)A-I applied to the AP surfaces of cells elicited BL ABCA1-mediated cholesterol efflux. Because ABCA1-mediated cholesterol efflux requires the presence of a cholesterol acceptor, we hypothesized that GBEC synthesize and secrete endogenous apo into the BL compartment. Here, we demonstrate that cholesterol loading of cells with model bile and AP apoA-I treatment is associated with an increase in the synthesis of apoE mRNA and protein. Furthermore, apoE is secreted into the BL compartment. LXRalpha/RXR ligands stimulate the synthesis of endogenous apoA-I mRNA and protein, as well as apoE mRNA. BL secretion of apoA-I is elicited by LXRalpha/RXR ligands. Therefore, GBEC synthesize apoA-I and -E and efflux cholesterol using ABCA1- and non-ABCA1- mediated pathways. These processes may alter gallbladder biliary cholesterol concentrations and thereby influence gallstone formation.     

ABCA1 increases extracellular ATP to mediate cholesterol efflux to ApoA-I

ATP-binding cassette protein A1 (ABCA1) is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apolipoprotein A-I (apoA-I). This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e., channel, pump, or flippase, remains unknown. In this study we analyzed extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of nonexpressing cells. We found that extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as wild-type ABCA1, is unable to raise extracellular ATP concentration, which suggests a casual relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e., <μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane-bound ectonucleotidase, CD39, abolishes cholesterol efflux to apoA-I. On the basis of these results, we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.     

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.     

Lysine residues of ABCA1 are required for the interaction with apoA-I

ATP-binding cassette protein A1 (ABCA1) plays a pivotal role in cholesterol homeostasis by generating high-density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), a lipid acceptor for ABCA1, reportedly interacts with ABCA1. However, it has also been proposed that apoA-I interacts with ABCA1-generated special domains on the plasma membrane, but apart from ABCA1, and solubilizes membrane lipids. To determine the importance of the apoA-I-ABCA1 interaction in HDL formation, the electrostatic interaction between apoA-I and ABCA1, which mediates the interaction between apoB100 in low-density lipoprotein particles (LDL) and LDL receptor, was analyzed. The apoA-I binding to ABCA1 and the cross-linking between them were inhibited by the highly charged molecules heparin and poly-L-lysine. Treating cells with membrane impermeable reagents that specifically react with primary amino groups abolished the interaction between apoA-I and ABCA1. However, these reagents did not affect the characteristic tight ATP binding to ABCA1. These results suggest that lysine residues in the extracellular domains of ABCA1 contribute to the interaction with apoA-I. The electrostatic interaction between ABCA1 and apoA-I is predicted to be the first step in HDL formation. This article is part of a Special Issue entitled Advances in high density lipoprotein formation and metabolism: a tribute to John F. Oram (1945-2010).     

Lipidation of apolipoprotein A-I by ATP-binding cassette transporter (ABC) A1 generates an interaction partner for ABCG1 but not for scavenger receptor BI

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL(1:40)) or 1:100 (rHDL(1:100)). Knock-down of ABCA1 inhibits the cellular binding at 4 degrees C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL(1:40) whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL(1:100). Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 degrees C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-beta- to alpha-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

Correction of apolipoprotein A-I-mediated lipid efflux and high density lipoprotein particle formation in human Niemann-Pick type C disease fibroblasts

Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of low plasma HDL-cholesterol in humans. We show here that treatment of human NPC1(-/-) fibroblasts with the liver X receptor (LXR) agonist TO-901317 increases ABCA1 expression and activity in human NPC1(-/-) fibroblasts, as indicated by near normalization of efflux of radiolabeled phosphatidylcholine and a marked increase in efflux of cholesterol mass to apoA-I. LXR agonist treatment prior to and during apoA-I incubation resulted in reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes, as well as cholesterol mass, in NPC1(-/-) cells. HDL species in human NPC disease plasma showed the same pattern of diminished large, cholesterol-rich alpha-1 HDL particles as seen in isolated heterozygous ABCA1 deficiency. Incubating NPC1(-/-) fibroblasts with the LXR agonist normalized the pattern of HDL particle formation by these cells. ABCG1, another LXR target gene involved in cholesterol efflux to HDL, also showed diminished expression in NPC1(-/-) fibroblasts and increased expression upon LXR agonist treatment. These results suggest that NPC1 mutations can be largely bypassed and that NPC1 protein function is non-essential for the trafficking and removal of cellular cholesterol if the down-stream defects in ABCA1 and ABCG1 regulation in NPC disease cells are corrected using an LXR agonist.     

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

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