Purification and ATPase activity of human ABCA1

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.     

ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.     

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.     

Highly purified scavenger receptor class B,type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI is a high and low density lipoprotein (HDL and LDL) receptor that mediates selective uptake of cholesteryl esters. Here we describe a reconstituted phospholipid/cholesterol liposome assay of the binding and selective uptake activities of SR-BI derived from detergent-solubilized cells. The assay, employing lysates from epitope-tagged receptor (mSR-BI-t1)-expressing mammalian and insect cells, recapitulated many features of SR-BI activity in intact cells, including high affinity and saturable (125)I-HDL binding, selective lipid uptake from [(3)H]cholesteryl ether-labeled HDL, and poor inhibition of HDL receptor activity by LDL. The novel properties of a mutated receptor (Q402R/Q418R, normal LDL binding but loss of most HDL binding) were reproduced in the assay, as was the ability of the SR-BI homologue CD36 to bind HDL but not mediate efficient lipid uptake. In this assay, essentially homogeneously pure mSR-BI-t1, prepared by single-step immunoaffinity chromatography, mediated high affinity HDL binding and efficient selective lipid uptake from HDL. Thus, SR-BI-mediated HDL binding and selective lipid uptake are intrinsic properties of the receptor that do not require the intervention of other proteins or specific cellular structures or compartments.     

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.     

Cholesterol transport via ABCA1: New insights from solid-phase binding assay

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).     

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.     

Membrane features and activity of GPI-anchored enzymes: alkaline phosphatase reconstituted in model membranes

The influence of membrane lipid environment on the activity of GPI-anchored enzymes was investigated with human placental alkaline phosphatase reconstituted by a detergent-dialysis technique in liposomes composed of palmitoyloleoylphosphatidylcholine, alone or in mixture with lipids enriched along with the protein within lipid rafts: cholesterol, sphingomyelin, and GM1 ganglioside. The highest V max was recorded for a phosphatidylcholine/10% GM1 mixture (143 +/- 5 nmol of substrate hydrolyzed per minute per microgram of protein), while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes (M:M:M) (57 +/- 3 and 52 +/- 3, respectively). No significant differences in K m were detected. The protein segregation, assessed using the chemical cross-linker bis(sulfosuccinimidyl)suberate, increased with the protein:lipid ratio, within the 1:1200-1:4800 protein:lipid molar ratio range, but did not affect enzyme activity. The activity decreased when the order of the lipid bilayers was increased, higher for those containing cholesterol, as judged by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Finally, the GPI-enzyme activity was affected by membrane curvature. This result was suggested by a strong inverse correlation (Pearson's correlation coefficient = 0.91; p < 0.0001) between activity and liposome diameter, measured by laser light scattering and ranging between 59 +/- 6 nm for a phosphatidylcholine/10% GM1 mixture (displaying the highest activity) and 188 +/- 25 nm for a phosphatidylcholine/30% cholesterol mixture and 185 +/- 23 nm for raft-mimicking liposomes (displaying the lowest activities). The activity-membrane curvature relationship was further confirmed by comparing the activity of proteoliposomes having different sizes but identical lipid compositions. These data open the possibility that the activity of GPI-anchored enzymes may be modulated by membrane microenvironment features, in particular by membrane curvature and cholesterol-enriched ordered microenvironments, such as those of lipid rafts.     

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.     

Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1

Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1?     

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.     

Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.     

ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation

Here we demonstrate that the ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. Targeted disruption of Abcg1 in mice has no effect on plasma lipids but results in massive accumulation of both neutral lipids and phospholipids in hepatocytes and in macrophages within multiple tissues following administration of a high-fat and -cholesterol diet. In contrast, overexpression of human ABCG1 protects murine tissues from dietary fat-induced lipid accumulation. Finally, we show that cholesterol efflux to HDL specifically requires ABCG1, whereas efflux to apoA1 requires ABCA1. These studies identify Abcg1 as a key gene involved in both cholesterol efflux to HDL and in tissue lipid homeostasis.     

Caveolin-1 interacts with ATP binding cassette transporter G1 (ABCG1) and regulates ABCG1-mediated cholesterol efflux

ATP-binding cassette transporter G1 (ABCG1) plays an important role in macrophage reverse cholesterol transport in vivo by promoting cholesterol efflux onto lipidated apoA-I. However, the underlying mechanism is unclear. Here, we found that ABCG1 co-immunoprecipitated with caveolin-1 (CAV1) but not with flotillin-1 and -2. Knockdown of CAV1 expression using siRNAs significantly reduced ABCG1-mediated cholesterol efflux without detectable effect on ABCA1-mediated cholesterol efflux. Disruption of the putative CAV1 binding site in ABCG1, through replacement of tyrosine residues at positions 487 and 489 or at positions 494 and 495 with alanine (Y487AY489A and Y494AY495A), impaired the interaction of ABCG1 with CAV1 and significantly decreased ABCG1-mediated cholesterol efflux. The substitution of Tyr494 and Tyr495 with Phe or Trp that resulted in an intact CAV1 binding site had no effect. Furthermore, Y494AY495A affected trafficking of ABCG1 to the cell surface. The mutant protein is mainly located intracellularly. Finally, we found that CAV1 co-immunoprecipitated with ABCG1 and regulated cholesterol efflux to reconstituted HDL in THP-1-derived macrophages upon the liver X receptor agonist treatment. These findings indicate that CAV1 interacts with ABCG1 and regulates ABCG1-mediated cholesterol efflux.     

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.     

Sphingomyelin-dependence of cholesterol efflux mediated by ABCG1

ABCG1, one of the half-type ATP binding cassette (ABC) proteins, mediates the efflux of cholesterol to HDL and functions in the reverse cholesterol transport from peripheral cells to the liver. We have shown that ABCG1 mediates the efflux of not only cholesterol but also sphingomyelin (SM) and phosphatidylcholine. Because SM preferentially associates with cholesterol, we examined whether it plays an important role in the ABCG1-mediated efflux of cholesterol. The efflux of cholesterol and SM mediated by ABCG1 was reduced in a mutant CHO-K1 cell line, LY-A, in which the cellular SM level is reduced because of a mutation of the ceramide transfer protein CERT. In contrast, CHO-K1 cells overexpressing CERT showed an increased efflux of cholesterol and SM mediated by ABCG1. The sensitivity of cells to methyl-beta-cyclodextrin suggested that cholesterol in nonraft domains was increased due to the disruption of raft domains in LY-A cells. These results suggest that the ABCG1-mediated efflux of cholesterol and SM is dependent on the cellular SM level and distribution of cholesterol in the plasma membrane.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

Structure of the Human Cholesterol Transporter ABCG1

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.     

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE^−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.