ABCMdb

ABCA1 p.Trp590Ser

ClinVar:	c.1769G>C , p.Trp590Ser D , Pathogenic
Predicted by SNAP2:	A: D (71%), C: D (71%), D: D (85%), E: D (80%), F: D (71%), G: D (80%), H: D (85%), I: D (75%), K: D (80%), L: D (80%), M: D (80%), N: D (75%), P: D (85%), Q: D (75%), R: D (75%), S: D (75%), T: D (71%), V: D (75%), Y: D (66%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]

Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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No. Sentence Comment
155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment

[hide] ABC proteins protect the human body and maintain o... Biosci Biotechnol Biochem. 2011;75(3):401-9. Epub 2011 Mar 7. Ueda K
ABC proteins protect the human body and maintain optimal health.
Biosci Biotechnol Biochem. 2011;75(3):401-9. Epub 2011 Mar 7., [PMID:21389634]

Abstract [show]
Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

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96 The Tangier mutation W590S, with one amino acid substitution in the first ECD, impairs HDL formation.64,66) Nagao analyzed the function of this Tangier mutant in detail, and found that it greatly decreases NaTC-dependent cholesterol and PC efflux.87) The kinetics of apoA-I binding to cells expressing ABCA1- W590S were similar to those for cells expressing wild-type ABCA1, consistently with a previous report that the W590S mutation does not impair apoA-I binding,64,65,88,89) but the W590S mutation delayed the dissociation of apoA-I from ABCA1.87) Based on these results, we proposed a four-step model for ABCA1-mediated HDL formation (Fig. 4),90) at the apoA-I binding step, ATP binding and/or hydrolysis causes conformational changes within the ECDs of ABCA1, to which apoA-I directly binds; at the translocation step, lipid translocation by ABCA1, which is apoA-I-independent occurs; at the loading step, lipid loading of apoA-I bound to ABCA1 occurs; and at the dissociation step, dissociation of lipid-loaded apoA-I from ABCA1 occurs. Login to comment
98 ApoA-I undergoes a conformational transition in response to lipids,91) and lipidated apoA-I does not interact with ABCA1.82,92) Because the W590S mutation impairs the translocation step, apoA-I can remain in lipid-free conformation, which has high affinity for ABCA1. Login to comment

[hide] Tweaking the cholesterol efflux capacity of recons... Biochem Cell Biol. 2012 Oct;90(5):636-45. doi: 10.1139/o2012-015. Epub 2012 May 18. Ma CI, Beckstead JA, Thompson A, Hafiane A, Wang RH, Ryan RO, Kiss RS
Tweaking the cholesterol efflux capacity of reconstituted HDL.
Biochem Cell Biol. 2012 Oct;90(5):636-45. doi: 10.1139/o2012-015. Epub 2012 May 18., [PMID:22607224]

Abstract [show]
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.

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6 Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. Login to comment
17 L`efflux de rHDL contenant de la PC était stimulé par la transfection d`un mutant d`ABCA1 non fonctionnel (W590S), suggérant que la liaison à ABCA1 représente une interaction compétitive. Login to comment
67 BHK cells overexpressing ABCG1, ABCA1, and a nonfunctional mutant, W590S ABCA1, were generated using mifepristone-inducible GeneSwitch system as previously described (Vaughan and Oram 2005, 2006). Login to comment
113 Thus, as a negative control, we included BHK cells that inducibly express a nonfunctional mutant ABCA1 (W590S). Login to comment
140 Efflux assays were performed with the four rHDL (5 mg panel A; 10 mg panel B) in the different cholesterol loaded BHK cells: BHK cells with no expressed protein (WT); BHK cells expressing ABCA1 (ABCA1); BHK cells expressing a dominant negative mutant of ABCA1 (ABCA1 W590S); and BHK cells expressing ABCG1 (ABCG1). Login to comment
146 Secondly, for WT-PC rHDL, a demonstrable increase in binding to ABCA1 and ABCG1-expressing cells was observed, with no increase in binding to cells expressing the nonfunctional W590S ABCA1, compared with control BHK cells. Login to comment
149 Thirdly, for WT-SM rHDL, we observed a moderate decrease in binding to ABCA1-expressing cells, a moderate increase in binding to ABCA1 W590S-expressing cells, and a sizeable and significant increase in binding to ABCG1-expressing cells. Login to comment
170 Binding assays were performed with the four radiolabeled rHDL (10 mg apoA-I protein) on BHK cells with no expressed protein (WT), BHK cells expressing ABCA1 (ABCA1), BHK cells expressing a nonfunctional mutant ABCA1 (ABCA1 W590S), and BHK cells expressing ABCG1 (ABCG1). Login to comment
198 However, rHDL with SM does not interact with ABCA1 (compare the efflux and binding for each rHDL with ABCA1 and the nonfunctional ABCA1 W590S). Login to comment

[hide] The interaction of ApoA-I and ABCA1 triggers signa... Mol Med. 2012 Mar 27;18(1):149-58. doi: 10.2119/molmed.2011.00183. Zhao GJ, Yin K, Fu YC, Tang CK
The interaction of ApoA-I and ABCA1 triggers signal transduction pathways to mediate efflux of cellular lipids.
Mol Med. 2012 Mar 27;18(1):149-58. doi: 10.2119/molmed.2011.00183., [PMID:22064972]

Abstract [show]
Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca(2)(+), and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.

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43 ApoA-I failed to increase ABCA1-W590S levels in these studies, suggesting that apoA-I binding was not sufficient for ABCA1 stabilization (36). Login to comment
45 Another interpretation is that apoA-I fails to bind the W590S mutant in the correct orientation, and therefore the appropriate conformational change of ABCA1 required to decrease calpain proteolysis does not occur. Login to comment
194 This appears to be a necessary, but not sufficient, step for lipid efflux (88), because, in the case of the ABCA1 mutant (Trp590Ser) that is associated with Tangier disease, an apoA-I/ABCA1 complex also is formed, but the apoA-I is not lipidated (15). Login to comment

[hide] Regulation of ABCA1 functions by signaling pathway... Biochim Biophys Acta. 2012 Mar;1821(3):522-9. doi: 10.1016/j.bbalip.2011.08.015. Epub 2011 Sep 5. Liu Y, Tang C
Regulation of ABCA1 functions by signaling pathways.
Biochim Biophys Acta. 2012 Mar;1821(3):522-9. doi: 10.1016/j.bbalip.2011.08.015. Epub 2011 Sep 5., [PMID:21920460]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1) is an integral cell membrane protein that protects cardiovascular disease by at least two mechanisms: by export of excess cholesterol from cells and by suppression of inflammation. ABCA1 exports cholesterol and phospholipids from cells by multiple steps that involve forming cell surface lipid domains, binding of apolipoproteins to ABCA1, activating signaling pathways, and solubilizing these lipids by apolipoproteins. ABCA1 executes its anti-inflammatory effect by modifying cell membrane lipid rafts and directly activating signaling pathways. The interaction of apolipoproteins with ABCA1 activates multiple signaling pathways, including Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), protein kinase A, Rho family G protein CDC42 and protein kinase C. Activating protein kinase A and Rho family G protein CDC42 regulates ABCA1-mediated lipid efflux, activating PKC stabilizes ABCA1 protein, and activating JAK2/STAT3 regulates both ABCA1-mediated lipid efflux and anti-inflammation. Thus, ABCA1 behaves both as a lipid exporter and a signaling receptor. Targeting ABCA1 receptor-like property using agonists for ABCA1 protein could become a promising new therapeutic target for increasing ABCA1 function and treating cardiovascular disease. 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).

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58 However, the analysis of naturally-occurring ABCA1 mutations indicates that the solubilization and release of apoA-I with lipids may not be a passive process, as the missense mutation W590S, which is still able to bind and release apoA-I back into the media, has a low capacity to transfer lipids to the released apoA-I [47,48,52]. Login to comment

[hide] Function and regulation of ABCA1--membrane meso-do... FEBS J. 2011 Sep;278(18):3190-203. doi: 10.1111/j.1742-4658.2011.08170.x. Epub 2011 Jun 13. Nagao K, Tomioka M, Ueda K
Function and regulation of ABCA1--membrane meso-domain organization and reorganization.
FEBS J. 2011 Sep;278(18):3190-203. doi: 10.1111/j.1742-4658.2011.08170.x. Epub 2011 Jun 13., [PMID:21554545]

Abstract [show]
The ATP-binding cassette protein A1 (ABCA1) mediates the secretion of cellular-free cholesterol and phospholipids to an extracellular acceptor, apolipoprotein A-I, to form high-density lipoprotein. Because ABCA1 is a key factor in cholesterol homeostasis, elaborate transcriptional and post-transcriptional regulations of ABCA1 have evolved to maintain cholesterol homeostasis. Recent studies suggest that ABCA1 moves lipids not only between membranes but also within membranes to organize and reorganize membrane meso-domains to modulate cell proliferation and immunity.

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82 The third group is the 'lipid translocation defect mutant`, represented by the W590S mutation in the first ECD of ABCA1 (Table 2). Login to comment
83 The subcellular distribution of W590S is indistinguishable from that of wild-type ABCA1. Login to comment
84 Furthermore, W590S mutation does not affect apoA-I binding [9,10,49,50]; however, W590S mutation reduced phosphatidylserine (PS) flopping activity of ABCA1, detected with the annexin V binding assay [49], and impaired sodium taurocholate-dependent cholesterol and phospholipid efflux by ABCA1 [51]. Login to comment
85 These results suggest that W590S mutation affects the lipid translocation activity of ABCA1 and that the two activities of ABCA1 (apoA-I binding and lipid translocation) can be separable (Fig. 3A). Login to comment
86 Additionally, W590S mutation retarded the dissociation of apoA-I from ABCA1 [51]. Login to comment

[hide] ABC transporters, atherosclerosis and inflammation... Atherosclerosis. 2010 Aug;211(2):361-70. Epub 2010 Jan 21. Fitzgerald ML, Mujawar Z, Tamehiro N
ABC transporters, atherosclerosis and inflammation.
Atherosclerosis. 2010 Aug;211(2):361-70. Epub 2010 Jan 21., [PMID:20138281]

Abstract [show]
Atherosclerosis, driven by inflamed lipid-laden lesions, can occlude the coronary arteries and lead to myocardial infarction. This chronic disease is a major and expensive health burden. However, the body is able to mobilize and excrete cholesterol and other lipids, thus preventing atherosclerosis by a process termed reverse cholesterol transport (RCT). Insight into the mechanism of RCT has been gained by the study of two rare syndromes caused by the mutation of ABC transporter loci. In Tangier disease, loss of ABCA1 prevents cells from exporting cholesterol and phospholipid, thus resulting in the build-up of cholesterol in the peripheral tissues and a loss of circulating HDL. Consistent with HDL being an athero-protective particle, Tangier patients are more prone to develop atherosclerosis. Likewise, sitosterolemia is another inherited syndrome associated with premature atherosclerosis. Here mutations in either the ABCG5 or G8 loci, prevents hepatocytes and enterocytes from excreting cholesterol and plant sterols, including sitosterol, into the bile and intestinal lumen. Thus, ABCG5 and G8, which from a heterodimer, constitute a transporter that excretes cholesterol and dietary sterols back into the gut, while ABCA1 functions to export excess cell cholesterol and phospholipid during the biogenesis of HDL. Interestingly, a third protein, ABCG1, that has been shown to have anti-atherosclerotic activity in mice, may also act to transfer cholesterol to mature HDL particles. Here we review the relationship between the lipid transport activities of these proteins and their anti-atherosclerotic effect, particularly how they may reduce inflammatory signaling pathways. Of particular interest are recent reports that indicate both ABCA1 and ABCG1 modulate cell surface cholesterol levels and inhibit its partitioning into lipid rafts. Given lipid rafts may provide platforms for innate immune receptors to respond to inflammatory signals, it follows that loss of ABCA1 and ABCG1 by increasing raft content will increase signaling through these receptors, as has been experimentally demonstrated. Moreover, additional reports indicate ABCA1, and possibly SR-BI, another HDL receptor, may directly act as anti-inflammatory receptors independent of their lipid transport activities. Finally, we give an update on the progress and pitfalls of therapeutic approaches that seek to stimulate the flux of lipids through the RCT pathway.

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1587 The exception to this behavior is the activity of the ABCA1 (W590S) missense mutant that is still able to bind and release apoA-I back into the media but fails to transfer lipid to the released apoA-I [24,25,28,29]. Login to comment

[hide] A novel missense mutation of ABCA1 in transmembran... Atherosclerosis. 2009 Sep;206(1):216-22. Epub 2009 Feb 25. Maekawa M, Kikuchi J, Kotani K, Nagao K, Odgerel T, Ueda K, Kawano M, Furukawa Y, Sakurabayashi I
A novel missense mutation of ABCA1 in transmembrane alpha-helix in a Japanese patient with Tangier disease.
Atherosclerosis. 2009 Sep;206(1):216-22. Epub 2009 Feb 25., [PMID:19344898]

Abstract [show]
Tangier disease (TD) is a hereditary disorder characterized by the severe deficiency or absence of high-density lipoprotein cholesterol (HDL-C). TD is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene, most of which are located in the extracellular loops and nucleotide-binding domains. Here we describe the first case of TD carrying a missense mutation in a transmembrane alpha-helix of ABCA1. A 31-year-old Japanese woman had an extremely low level of HDL-C (1mg/dl) and yellowish tonsillar swelling, leading to the diagnosis of TD. The proband was homozygous for a point mutation of T4978C in exon 37, which results in the substitution of cysteine-1660 to arginine (C1660R) in the 8th transmembrane segment of ABCA1. Her parents, grandmother, and brother were found to be heterozygous for the same mutation. Both peripheral blood leukocytes from the patient and HEK293 cells transfected with T4978C-mutated ABCA1 normally expressed ABCA1 on the plasma membrane and had normal apolipoprotein A-I-binding ability. However, apolipoprotein A-I-mediated efflux of cholesterol and phospholipids was markedly diminished in HEK293 cells transfected with T4978C-mutated ABCA1. These results suggest that this mutant is normally translated and exists as a stable product with normal localization, yet is functionally defective. Cysteine-1660 appears to be a critical residue for cholesterol transport of ABCA1.

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152 The loss of lipid efflux without impaired apo A-I binding was also observed in A255T, W590S, and T929I mutations [18]. Login to comment
153 Surface binding of apo A-I was intact in W590S mutant-transfected HeLa and HEK293 cells [18,26], and direct binding of apo A-I was enhanced in the W590S mutant compared with wild-type ABCA1 in chemical cross-linking studies [27,28]. Login to comment
155 Rigot et al. [26] reported that the reduction of plasma membrane flipping might underlie the loss of lipid efflux in the W590S mutant. Login to comment

[hide] Sodium taurocholate-dependent lipid efflux by ABCA... J Lipid Res. 2009 Jun;50(6):1165-72. Epub 2009 Feb 8. Nagao K, Zhao Y, Takahashi K, Kimura Y, Ueda K
Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation.
J Lipid Res. 2009 Jun;50(6):1165-72. Epub 2009 Feb 8., [PMID:19202195]

Abstract [show]
ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.

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0 Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation Kohjiro Nagao,* Yu Zhao,* Kei Takahashi,* Yasuhisa Kimura,* and Kazumitsu Ueda1,*,† * Laboratory of Cellular Biochemistry,* Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan; and Institute for Integrated Cell-Material Sciences,† Kyoto University, Kyoto 606-8502, Japan Abstract ABCA1 plays a major role in HDL metabolism. Login to comment
6 The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment
7 However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. Login to comment
8 These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. Login to comment
9 NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.- Nagao, K., Y. Zhao, K. Takahashi, Y. Kimura, and K. Ueda. Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation. Login to comment
45 Cell culture Human embryonic kidney (HEK293) cells and WI-38 fibroblasts were grown in a humidified incubator (5% CO2) at 37°C in DMEM supplemented with 10% heat-inactivated FBS. Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment
101 The W590S Tangier mutation impairs NaTC-dependent lipid efflux but not apoA-I binding It has been reported that ABCA1 carrying the Tangier disease mutation W590S in the first extra cellular domain is correctly targeted to the plasma membrane and interacts with apoA-I but fails to mediate normal apoA-I-dependent lipid efflux (3, 26-28). Login to comment
102 We compared NaTC-dependent lipid efflux from HEK293 cells stably expressing wild-type or ABCA1-W590S fused to GFP at the C terminus. Login to comment
103 Western blotting (Fig. 4A) and fluorescence microscopy (see supplementary Fig. II) suggested that the expression and surface localization of the wild type and ABCA1-W590S were similar. Login to comment
104 Both NaTC-dependent and apoA-I-dependent cholesterol and phospholipid efflux were greatly decreased by the W590S mutation (Fig. 4B, C). Login to comment
105 As previously reported, apoA-I bound to the cell surface expressing ABCA1-W590S as efficiently as to that expressing wild-type ABCA1 (see supplementary Fig. II). Login to comment
119 1168 Journal of Lipid Research Volume 50, 2009 atHealthScienceLibraryCB#7585,onSeptember24,2012www.jlr.orgDownloadedfrom 0.DC1.html are mediated by ABCA1 in an ATP-dependent manner, are in fact separable and that the W590S mutation impairs only the latter step. Login to comment
124 The W590S mutation also impaired cholesterol secretion at 27°C. Login to comment
127 The kinetics of apoA-I binding to cells expressing ABCA1-W590S was quite similar to that to cells expressing wild-type ABCA1. Login to comment
129 In contrast, ,40% of apoA-I dissociated from cells expressing ABCA1-W590S in 2 min, and ?60% of apoA-I dissociated in 4 min. Login to comment
130 These results suggest that the W590S mutation retards apoA-I dissociation from cells. Login to comment
138 The W590S mutation impairs NaTC-dependent lipid efflux by ABCA1. Login to comment
141 The efflux of cellular free cholesterol (B) and choline phospholipids (C) was analyzed. HEK/ABCA1-GFP cells and HEK/ ABCA1-W590S-GFP cells were incubated for 24 h in DMEM containing 0.02% BSA (open bars), 0.02% BSA and 1 mM NaTC (filled bars), or 0.02% BSA and 5 mg/ml apoA-I (hatched bars). Login to comment
144 HEK/ABCA1-GFP, HEK/ABCA1-MM-GFP, or HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for the indicated times at 27°C. Login to comment
154 There may be other proteins (32, 33) in WI-38 cells that mediate cholesterol efflux in the presence of apoA-I. Alternatively, binding of significant amounts of apoA-I on the cell surface may cause spontaneous cholesterol loading on apoA-I. Indeed, greater cholesterol efflux was observed in the presence of apoA-I from cells expressing ABCA1-W590S, which allows apoA-I binding, than in the presence of NaTC, and this effect was strongly suppressed at 27°C (Fig. 4; see supplementary Fig. VI). Login to comment
161 The Tangier mutation W590S, which impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment
162 It has been reported that ABCA1-W590S interacts in a normal manner with ATP (3). Login to comment
163 The W590S mutation may abolish the coupling of ATP-induced conformational changes of ABCA1 with lipid translocation. Login to comment
164 The kinetics of apoA-I binding to cells expressing ABCA1-W590S were similar to those of binding to cells expressing wild-type ABCA1, consistent with a previous report showing that the W590S mutation does not impair apoA-I binding (8, 26-28). Login to comment
165 Thus, the W590S mutation would not affect the conformational changes of ABCA1 Fig. 7. Login to comment
173 Time-dependent apoA-I dissociation from cells expressing ABCA1 or ABCA1-W590S. Login to comment
174 HEK/ABCA1-GFP and HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for 15 min at 37°C and further incubated with 25 mg/ml nonlabeled apoA-I for the indicated times at 27°C. Login to comment
178 The W590S mutation retarded the dissociation of apoA-I from ABCA1. Login to comment
186 In addition, ABCA1-W590S was reported not to be stabilized by apoA-I (29). Login to comment
189 Our results suggest that the W590S mutation impaired NaTC-dependent lipid efflux but not apoA-I binding. Login to comment
191 These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading facilitates apoA-I dissociation from ABCA1. Login to comment
5 The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment
43 Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment
96 The W590S Tangier mutation impairs NaTC-dependent lipid efflux but not apoA-I binding It has been reported that ABCA1 carrying the Tangier disease mutation W590S in the first extra cellular domain is correctly targeted to the plasma membrane and interacts with apoA-I but fails to mediate normal apoA-I-dependent lipid efflux (3, 26-28). Login to comment
97 We compared NaTC-dependent lipid efflux from HEK293 cells stably expressing wild-type or ABCA1-W590S fused to GFP at the C terminus. Login to comment
98 Western blotting (Fig. 4A) and fluorescence microscopy (see supplementary Fig. II) suggested that the expression and surface localization of the wild type and ABCA1-W590S were similar. Login to comment
99 Both NaTC-dependent and apoA-I-dependent cholesterol and phospholipid efflux were greatly decreased by the W590S mutation (Fig. 4B, C). Login to comment
100 As previously reported, apoA-I bound to the cell surface expressing ABCA1-W590S as efficiently as to that expressing wild-type ABCA1 (see supplementary Fig. II). Login to comment
114 1168 Journal of Lipid Research Volume 50, 2009 at SEMMELWEIS EGYETEM NET KORELET, on December 3, 2015 www.jlr.org Downloaded from .DC1.html are mediated by ABCA1 in an ATP-dependent manner, are in fact separable and that the W590S mutation impairs only the latter step. Login to comment
122 The kinetics of apoA-I binding to cells expressing ABCA1-W590S was quite similar to that to cells expressing wild-type ABCA1. Login to comment
125 These results suggest that the W590S mutation retards apoA-I dissociation from cells. Login to comment
133 The W590S mutation impairs NaTC-dependent lipid efflux by ABCA1. Login to comment
136 The efflux of cellular free cholesterol (B) and choline phospholipids (C) was analyzed. HEK/ABCA1-GFP cells and HEK/ ABCA1-W590S-GFP cells were incubated for 24 h in DMEM containing 0.02% BSA (open bars), 0.02% BSA and 1 mM NaTC (filled bars), or 0.02% BSA and 5 mg/ml apoA-I (hatched bars). Login to comment
139 HEK/ABCA1-GFP, HEK/ABCA1-MM-GFP, or HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for the indicated times at 27&#b0;C. Login to comment
149 Alternatively, binding of significant amounts of apoA-I on the cell surface may cause spontaneous cholesterol loading on apoA-I. Indeed, greater cholesterol efflux was observed in the presence of apoA-I from cells expressing ABCA1-W590S, which allows apoA-I binding, than in the presence of NaTC, and this effect was strongly suppressed at 27&#b0;C (Fig. 4; see supplementary Fig. VI). Login to comment
156 The Tangier mutation W590S, which impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. Login to comment
157 It has been reported that ABCA1-W590S interacts in a normal manner with ATP (3). Login to comment
158 The W590S mutation may abolish the coupling of ATP-induced conformational changes of ABCA1 with lipid translocation. Login to comment
159 The kinetics of apoA-I binding to cells expressing ABCA1-W590S were similar to those of binding to cells expressing wild-type ABCA1, consistent with a previous report showing that the W590S mutation does not impair apoA-I binding (8, 26-28). Login to comment
160 Thus, the W590S mutation would not affect the conformational changes of ABCA1 Fig. 7. Login to comment
168 Time-dependent apoA-I dissociation from cells expressing ABCA1 or ABCA1-W590S. Login to comment
169 HEK/ABCA1-GFP and HEK/ABCA1-W590S-GFP cells were incubated with 5 mg/ml Alexa546-labeled apoA-I for 15 min at 37&#b0;C and further incubated with 25 mg/ml nonlabeled apoA-I for the indicated times at 27&#b0;C. Login to comment
181 In addition, ABCA1-W590S was reported not to be stabilized by apoA-I (29). Login to comment
184 Our results suggest that the W590S mutation impaired NaTC-dependent lipid efflux but not apoA-I binding. Login to comment

[hide] Formation of two intramolecular disulfide bonds is... J Biol Chem. 2009 Apr 24;284(17):11293-300. Epub 2009 Mar 3. Hozoji M, Kimura Y, Kioka N, Ueda K
Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.
J Biol Chem. 2009 Apr 24;284(17):11293-300. Epub 2009 Mar 3., [PMID:19258317]

Abstract [show]
ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys(75) and Cys(309)) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys(1477) was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys(1463) and Cys(1465), impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys(75), Cys(309), Cys(1463), Cys(1465), and Cys(1477)) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys(75) and Cys(309) in ECD1 and either Cys(1463) or Cys(1465) and Cys(1477) in ECD2) are required for ABCA1 to be fully functional.

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192 The Tangier mutation W590S impairs apoA-I-dependent cholesterol efflux but not apoA-I binding (15). Login to comment
193 The replacement of C15 by serine may cause conformational changes similar to those caused by the W590S mutation. Login to comment
190 The Tangier mutation W590S impairs apoA-I-dependent cholesterol efflux but not apoA-I binding (15). Login to comment
191 The replacement of C15 by serine may cause conformational changes similar to those caused by the W590S mutation. Login to comment

[hide] Retroendocytosis pathway of ABCA1/apoA-I contribut... Genes Cells. 2009 Feb;14(2):191-204. Epub 2008 Jan 6. Azuma Y, Takada M, Shin HW, Kioka N, Nakayama K, Ueda K
Retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation.
Genes Cells. 2009 Feb;14(2):191-204. Epub 2008 Jan 6., [PMID:19170766]

Abstract [show]
ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.

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36 A Tangier disease-type mutation (W590S) greatly reduced the cholesterol efflux activity of ABCA1(207HA), and lysine-to-methionine mutations (MM) in both of the two ATP-binding domains almost completely abolished activity (Fig. 1E), as we previously reported in the context of ABCA1 or ABCA1-GFP (Tanaka et al. 2003). Login to comment
37 As shown in Fig. S1 in Supporting Information/Supplementary material, the subcellular localization and cell surface expression of the W590S and MM mutants of ABCA1(207HA) (lower panels) were indistinguishable from those of the corresponding mutants of ABCA1-GFP. Login to comment
197 DNA construction We constructed plasmids for expressing human wild-type ABCA1, ABCA1(W590S) and ABCA1(K939M,K1952M)(MM) bearing an insertion of the influenza virus hemagglutinin (HA) epitope sequence between residues 207 and 208 (within the first extracellular loop), using the bicistronic expression vector pHaMAIRESneo. Login to comment

[hide] ABCA1 mutants reveal an interdependency between li... J Lipid Res. 2009 Feb;50(2):285-92. Epub 2008 Sep 5. Vaughan AM, Tang C, Oram JF
ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation.
J Lipid Res. 2009 Feb;50(2):285-92. Epub 2008 Sep 5., [PMID:18776170]

Abstract [show]
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.

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49 the first extracellular loop (V399A, R587W, W590S, and Q597R), two were in the second extracellular loop (C1477R and I1517R), and one was in the Walker A motif of the first nucleotide binding domain (A937V, NBD1) (Fig. 1). Login to comment
55 W590S.A1950V. Login to comment
82 This activation was nearly abolished in cells expressing all mutants except two (W590S and A1950V), which are the same two mutants that have the highest ability to cross-link apoA-I (Fig. 2E). Login to comment
96 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
113 Cholesterol and phospholipid efflux values for mutant W590S are labeled in panels A and B. Login to comment
126 A previous study showed that the W590S ABCA1 mutation severely impaired lipid efflux without affecting apoA-I cross-linking to ABCA1 (24), suggesting that apoA-I binding to ABCA1 can be dissociated from lipid export activity. Login to comment
129 The lipid export activity of the W590S ABCA1 mutant, however, was among the highest of the natural mutants, and thus values for cells expressing this mutant ABCA1 did not deviate far from the linear regression line when lipid transport activities were plotted against apoA-I/ABCA1 cross-linking (Fig. 4A, B). Login to comment
133 The two ABCA1 mutants that had near-normal apoA-I-induced JAK2 activation were W590S Fig. 6. Login to comment
95 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
112 Cholesterol and phospholipid efflux values for mutant W590S are labeled in panels A and B. Login to comment
125 A previous study showed that the W590S ABCA1 mutation severely impaired lipid efflux without affecting apoA-I cross-linking to ABCA1 (24), suggesting that apoA-I binding to ABCA1 can be dissociated from lipid export activity. Login to comment
128 The lipid export activity of the W590S ABCA1 mutant, however, was among the highest of the natural mutants, and thus values for cells expressing this mutant ABCA1 did not deviate far from the linear regression line when lipid transport activities were plotted against apoA-I/ABCA1 cross-linking (Fig. 4A, B). Login to comment
132 The two ABCA1 mutants that had near-normal apoA-I-induced JAK2 activation were W590S Fig. 6. Login to comment

[hide] The ABCA1 Q597R mutant undergoes trafficking from ... Biochem Biophys Res Commun. 2008 May 16;369(4):1174-8. Epub 2008 Mar 14. Tanaka AR, Kano F, Ueda K, Murata M
The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress.
Biochem Biophys Res Commun. 2008 May 16;369(4):1174-8. Epub 2008 Mar 14., [PMID:18343215]

Abstract [show]
Mutations in ATP binding cassette transporter 1 (ABCA1), a membrane protein associated with cellular cholesterol efflux, cause Tangier disease (TD). Previously, we showed that an ABCA1 Q597R mutant (QR) identified in TD is retained in the endoplasmic reticulum. Here, we report that QR trafficking to the plasma membrane was rapidly induced by thapsigargin or DTT, indicating that ER stress-induced QR trafficking. However, pharmacological rescue of ABCA1 activity was not observed. The trafficking was dependent on COPII components and occurred via the ER-Golgi intermediate compartments. Furthermore, we found that QR was more sensitive to ER stress than ATF6, a transcription factor associated with the ER stress response. These results suggest that thapsigargin can be effective in correcting trafficking defects, and raise the possibility that ER stress-induced trafficking is involved in ER quality control.

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116 Previously, we reported that the W590S mutant of ABCA1 shows functional defects, but is localized to the PM [17]. Login to comment
142 W590S mutation and the Q597R mutation are within extracellular domain 1 (the ER-lumenal domain) of ABCA1, which is a mutational hotspot in TD. Login to comment
115 Previously, we reported that the W590S mutant of ABCA1 shows functional defects, but is localized to the PM [17]. Login to comment
141 W590S mutation and the Q597R mutation are within extracellular domain 1 (the ER-lumenal domain) of ABCA1, which is a mutational hotspot in TD. Login to comment

[hide] The role of different regions of ATP-binding casse... Biochemistry. 2007 Aug 21;46(33):9388-98. Epub 2007 Jul 26. Mukhamedova N, Fu Y, Bukrinsky M, Remaley AT, Sviridov D
The role of different regions of ATP-binding cassette transporter A1 in cholesterol efflux.
Biochemistry. 2007 Aug 21;46(33):9388-98. Epub 2007 Jul 26., [PMID:17655203]

Abstract [show]
ABCA1 is a key element of cholesterol efflux, but the mechanism of ABCA1-dependent cholesterol efflux is still unclear. Monoclonal antibodies against ABCA1 were used to map functional domains of ABCA1. Two antibodies were directed against a fragment of the first extracellular loop of ABCA1, and the third antibody was directed against a fragment of the fourth extracellular loop. One antibody against the first loop inhibited cholesterol efflux from human macrophages without inhibiting apolipoprotein A-I (apoA-I) binding and internalization. Another antibody against the first loop inhibited apoA-I binding and internalization without inhibiting cholesterol efflux. The antibody against the fourth loop inhibited apoA-I binding to ABCA1 but enhanced cholesterol efflux from macrophages and reduced intracellular cholesterol content. This antibody also increased cholesterol efflux from HeLa cells transfected with ABCA1 but not from cells with DeltaPEST-ABCA1. The mechanism of the stimulating effect of this antibody on cholesterol efflux was found to be stabilization of ABCA1 leading to the increase in abundance of cell surface ABCA1. We conclude that a site on the first extracellular loop is required for cholesterol efflux, whereas a site on the fourth extracellular loop may be responsible for ABCA1 stability.

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269 Natural mutations found in the Tangier pedigree, R587W, W590S, Q597R, and S1506L, as well as generated mutant C1477R strongly inhibited cholesterol and phospholipid efflux (28, 35, 36) and, with the exception of W590S, also apoA-I binding (36). Login to comment
288 Other mutations (e.g., W590S) inhibited cholesterol efflux with apoA-I binding intact (30, 35, 36). Login to comment
315 As with the ABCA1 mutant W590S (35, 36), the antibody NDF4C2 inhibited cholesterol efflux without affecting apoA-I binding to ABCA1. Login to comment

[hide] Genetic etiology of isolated low HDL syndrome: inc... Arterioscler Thromb Vasc Biol. 2007 May;27(5):1139-45. Epub 2007 Feb 15. Kiss RS, Kavaslar N, Okuhira K, Freeman MW, Walter S, Milne RW, McPherson R, Marcel YL
Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.
Arterioscler Thromb Vasc Biol. 2007 May;27(5):1139-45. Epub 2007 Feb 15., [PMID:17303779]

Abstract [show]
OBJECTIVE: We have used a multitiered approach to identify genetic and cellular contributors to high-density lipoprotein (HDL) deficiency in 124 human subjects. METHODS AND RESULTS: We resequenced 4 candidate genes for HDL regulation and identified several functional nonsynonymous mutations including 2 in apolipoprotein A-I (APOA1), 4 in lecithin:cholesterol acyltransferase (LCAT), 1 in phospholipid transfer protein (PLTP), and 7 in the ATP-binding cassette transporter ABCA1, leaving 88% (110/124) of HDL deficient subjects without a genetic diagnosis. Cholesterol efflux assays performed using cholesterol-loaded monocyte-derived macrophages from the 124 low HDL subjects and 48 control subjects revealed that 33% (41/124) of low HDL subjects had low efflux, despite the fact that the majority of these subjects (34/41) were not carriers of dysfunctional ABCA1 alleles. In contrast, only 2% of control subjects presented with low efflux (1/48). In 3 families without ABCA1 mutations, efflux defects were found to cosegregate with low HDL. CONCLUSIONS: Efflux defects are frequent in low HDL syndromes, but the majority of HDL deficient subjects with cellular cholesterol efflux defects do not harbor ABCA1 mutations, suggesting that novel pathways contribute to this phenotype.

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47 In ABCA1, a total of 19 nonsynonymous coding sequence variants; some of these we reported previously.22 Of these, 9 sequence variants were common polymorphisms (ie, reported in the literature as common or of similar prevalence in control subjects): P85L, P85A, R219K, V399A, V771M, V825I, I883M, E1172D, R1587K.14,32-35 Another 5 sequence variants, identified here, were previously reported to be disease causing: W590L (reported as W590S)14; C1477F (reported as C1477R)13; S1731C (only found in French-Canadian populations)36; N1800H32; and 1851X.37 Five sequence variants were novel: K199F, H551D, R965C, E1386Q, and D1706N. Login to comment
42 In ABCA1, a total of 19 nonsynonymous coding sequence variants; some of these we reported previously.22 Of these, 9 sequence variants were common polymorphisms (ie, reported in the literature as common or of similar prevalence in control subjects): P85L, P85A, R219K, V399A, V771M, V825I, I883M, E1172D, R1587K.14,32-35 Another 5 sequence variants, identified here, were previously reported to be disease causing: W590L (reported as W590S)14; C1477F (reported as C1477R)13; S1731C (only found in French-Canadian populations)36; N1800H32; and 1851X.37 Five sequence variants were novel: K199F, H551D, R965C, E1386Q, and D1706N. Login to comment

[hide] ABCA1: at the nexus of cholesterol, HDL and athero... Trends Biochem Sci. 2007 Apr;32(4):172-9. Epub 2007 Feb 26. Attie AD
ABCA1: at the nexus of cholesterol, HDL and atherosclerosis.
Trends Biochem Sci. 2007 Apr;32(4):172-9. Epub 2007 Feb 26., [PMID:17324574]

Abstract [show]
Cholesterol is an essential component of eukaryotic membranes. To prevent the toxicity associated with cholesterol overload, cells transport excess cholesterol across the plasma membrane in part through the ABCA1 lipid transporter. The discovery that mutations in ABCA1 are associated with high-density lipoprotein (HDL)-deficiency syndromes led to studies that show ABCA1, through its transport of cholesterol and phospholipid to apolipoprotein acceptors in the bloodstream, is crucial for the formation of HDL particles. In the intestine, ABCA1 transports cholesterol from the epithelial cells to the bloodstream, contributing to approximately one-third of HDL production. In the arterial wall, excess cholesterol in macrophages is associated with atherosclerosis; here, ABCA1 is anti-atherogenic because it enables macrophages to rid themselves of excess cholesterol.

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142 This is supported by studies of the Trp590Ser ABCA1 mutant, which binds to apo-A1 but does not promote efficient cholesterol efflux and releases the apo-A1 in an unlipidated form [70,71]. Login to comment
155 This is also true of the Trp590Ser mutant mentioned earlier. Login to comment
156 Mutations in the ATP-binding modules of the Trp590Ser mutant prevent binding to apo-A1 [69], predicting that the multimeric form is the one bound by apo-A1. Login to comment

[hide] Specific mutations in ABCA1 have discrete effects ... Circ Res. 2006 Aug 18;99(4):389-97. Epub 2006 Jul 27. Singaraja RR, Visscher H, James ER, Chroni A, Coutinho JM, Brunham LR, Kang MH, Zannis VI, Chimini G, Hayden MR
Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro.
Circ Res. 2006 Aug 18;99(4):389-97. Epub 2006 Jul 27., [PMID:16873719]

Abstract [show]
Mutations in ATP-binding cassette transporter A1 (ABCA1) cause Tangier disease and familial hypoalphalipoproteinemia, resulting in low to absent plasma high-density lipoprotein cholesterol levels. However, wide variations in clinical lipid phenotypes are observed in patients with mutations in ABCA1. We hypothesized that the various lipid phenotypes would be the direct result of discrete and differing effects of the mutations on ABCA1 function. To determine whether there is a correlation between the mutations and the resulting phenotypes, we generated in vitro 15 missense mutations that have been described in patients with Tangier disease and familial hypoalphalipoproteinemia. Using localization of ABCA1, its ability to induce cell surface binding of apolipoprotein A-I, and its ability to elicit efflux of cholesterol and phospholipids to apolipoprotein A-I we determined that the phenotypes of patients correlate with the severity and nature of defects in ABCA1 function.

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43 In ABCA1 heterozygotes, 3 distinct phenotypic groups emerged, one in which HDL-C levels were Ϸ50% of those of age-and sex-matched controls, one in which HDL-C levels were at least 70% of controls (A255T, W590S, T929I), and one in which HDL-C levels were significantly below the expected 50% of the levels for controls (30.4% of controls) (M1091T) (Table). Login to comment
111 Three mutations fit this criteria, with patients harboring A255T showing 76%, W590S showing 83%, and T929I showing 76% of normal HDL-C levels. Login to comment
112 Intracellular Localization All 3 mutants A255T, W590S, and T929I, were localized by immunofluorescence to the plasma membrane and to intracellular regions in a manner indistinguishable from wild-type ABCA1 (Figure 5A). Login to comment
115 ApoA-I Binding All mutants showed normal ApoA-I binding compared with wild-type ABCA1 (A255T, 98.0Ϯ10.2%, nϭ4; W590S, 94.9Ϯ26.7%, nϭ3; T929I, 83.6Ϯ14.5, nϭ3) (Figure 5D). Login to comment
116 Cholesterol and Phosphocholine Efflux All 3 mutants displayed defects in both cholesterol (Figure 5E) and phosphocholine (Figure 5F) efflux (A255T, cholesterol 49.2Ϯ7.7%, nϭ5, Pϭ0.0001, choline 41.5Ϯ22.5%, nϭ8, Pϭ0.0002; W590S, cholesterol 47.1Ϯ13.1%, nϭ5, Pϭ0.0008, choline 44.7Ϯ21.1%, nϭ3, PϽ0.05; and T929I, Figure 4. Login to comment
125 Mutant W590S has been previously shown to induce reduced annexin V binding, which is a measure of plasma membrane flipping.18 Plasma membrane flipping is observed in wild-type ABCA1-expressing cells and may be a critical factor for efflux to occur. Login to comment
126 Recent work has shown that W590S associates normally with ApoA-I. However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free, indicating a defect in the lipidation of ApoA-I.24 When the ability of the ABCA1 mutants to promote ␣HDL formation was assessed (Figure 5G), wild-type ABCA1 was able to form ␣HDL of 10.4 to 12.2 nM diameter, whereas A255T and W590S formed only lipid-free ApoA-I and T929I formed lipid-free and ApoA-I of 7.1 to Ϸ9-nM diameter. Login to comment
132 Heterozygous patients with the mutations A255T, W590S and T929I show Ͼ70% of normal HDL-C levels, and therefore are hypothesized to have mutant alleles that partially retain function. Login to comment
135 C, Cell surface biotinylation revealed normal levels of A255T, W590S, and T929I at the plasma membrane. Login to comment
161 All 3 missense mutations (A255T, W590S, and T929I) that showed residual function were localized to the plasma membrane and induced cell surface ApoA-I binding at levels similar to wild-type ABCA1. Login to comment
162 Our findings for the mutant W590S therefore agree with previous findings that W590S does localize normally and shows normal ApoA-I binding.18,19,25 These data imply that although mutant ABCA1 localizes to the plasma membrane and induces ApoA-I binding, these events are insufficient for normal lipid efflux to occur. Login to comment
163 Previous studies showed that W590S, which has defective lipid efflux, cross-links efficiently to ApoA-I ,and its rate of dissociation from ApoA-I was similar to wild-type ABCA1.24 However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free.24 Mutants A255T, W590S, and T929I were normal in their binding to lipid-free ApoA-I. However, A255T and W590S failed completely to lipidate ApoA-I, and T929I produced lipidated species with abnormal size. Login to comment
164 One mutant, M1091T, when present in heterozygous patients, results in significantly lower than 50% of normal HDL-C and efflux levels. Login to comment
165 Previous studies showed that W590S, which has defective lipid efflux, cross-links efficiently to ApoA-I ,and its rate of dissociation from ApoA-I was similar to wild-type ABCA1.24 However, the ApoA-I released from wild-type ABCA1 was bound to lipids, whereas the ApoA-I released from W590S was lipid-free.24 Mutants A255T, W590S, and T929I were normal in their binding to lipid-free ApoA-I. However, A255T and W590S failed completely to lipidate ApoA-I, and T929I produced lipidated species with abnormal size. Login to comment

[hide] Transition from dimers to higher oligomeric forms ... J Biol Chem. 2006 Jul 21;281(29):20283-90. Epub 2006 May 18. Trompier D, Alibert M, Davanture S, Hamon Y, Pierres M, Chimini G
Transition from dimers to higher oligomeric forms occurs during the ATPase cycle of the ABCA1 transporter.
J Biol Chem. 2006 Jul 21;281(29):20283-90. Epub 2006 May 18., [PMID:16709568]

Abstract [show]
Fluorescence resonance energy transfer and native PAGE analytical techniques were employed to assess the quaternary structure of ABCA1, an ATP binding cassette transporter playing a crucial role in cellular lipid handling. These experimental approaches support the conclusion that ABCA1 is associated in dimeric structures that undergo transition into higher order structures, i.e. tetramers, during the ATP catalytic cycle. Our data hence underline molecular assembly as a crucial parameter in ABCA1 function and the advantage of native PAGE as analytical tool for intractable membrane proteins.

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128 We chose to analyze W590S and C1477R, two Tangier-associated mutations previously characterized and known to differentially affect the ABCA1-induced binding of apoA-I and annexin V (3, 32). Login to comment
133 A:D ratio < 2.5 A:D ratio > 2.5 N-ter 14.86 Ϯ 1.4 19.11 Ϯ 1.3 YABCA1/CABCA1 n ϭ 11 n ϭ 17 Nand C-ter 17.72 Ϯ 2.1 18.91 Ϯ 2.312 C Y ABCA1/ABCA1 Y C n ϭ 11 n ϭ 8 C-ter 11.74 Ϯ 0.85 24.00 Ϯ 1.001 ABCA1Y/ABCA1C n ϭ 29 n ϭ 21 W590S 11.79 Ϯ 1.230 26.54 Ϯ 1.004 W590SY/W590SC n ϭ 12 n ϭ 30 C1477R 13.70 Ϯ 0.7299 26.50 Ϯ 1.462 C1477RY/C1477RC n ϭ 27 n ϭ 22 ABCA1MM 11.83 Ϯ 1.048 26.59 Ϯ 1.591 ABCA1MMY/ABCA1MMC n ϭ 14 n ϭ 15 Oligomerization of the ABCA1 Transporter 20286 constructs supported dimerization since an efficiency of the energy transfer similar to that of wild type and similarly sensitive to variations in acceptor-to-donor ratios was detected (Fig. 4, A and B, and Table 1). Login to comment
134 However, upon fractionation on native PAGE, the lysates of cells transfected with W590S or C1477R showed a higher molecular mass band migrating at Ͼ800 kDa and accounting for 9 and 27%, respectively, of the total ABCA1 signal (Fig. 4C, upper panel), in the absence of any modification of expression levels as shown by the SDS-PAGE analysis (Fig. 4C, lower panel). Login to comment
180 A and B, imaging FRET of cells expressing appropriately tagged W590S or C1477R, two ABCA1 variants associated with Tangier disease and variably affecting the functional readouts associated with ABCA1 expression. Login to comment
182 C, upper panel, native PAGE profiles of lysates from cells transfected with ABCA1Y, W590S, W590SY, or C1477RY indicate that the two variants associated in dimers. Login to comment
127 We chose to analyze W590S and C1477R, two Tangier-associated mutations previously characterized and known to differentially affect the ABCA1-induced binding of apoA-I and annexin V (3, 32). Login to comment
132 A:D ratio < 2.5 A:D ratio > 2.5 N-ter 14.86 afe; 1.4 19.11 afe; 1.3 YABCA1/CABCA1 n afd; 11 n afd; 17 Nand C-ter 17.72 afe; 2.1 18.91 afe; 2.312 C Y ABCA1/ABCA1 Y C n afd; 11 n afd; 8 C-ter 11.74 afe; 0.85 24.00 afe; 1.001 ABCA1Y/ABCA1C n afd; 29 n afd; 21 W590S 11.79 afe; 1.230 26.54 afe; 1.004 W590SY/W590SC n afd; 12 n afd; 30 C1477R 13.70 afe; 0.7299 26.50 afe; 1.462 C1477RY/C1477RC n afd; 27 n afd; 22 ABCA1MM 11.83 afe; 1.048 26.59 afe; 1.591 ABCA1MMY/ABCA1MMC n afd; 14 n afd; 15 Oligomerization of the ABCA1 Transporter 20286 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 29ߦJULY 21, 2006 at SEMMELWEIS UNIV OF MEDICINE on December 3, constructs supported dimerization since an efficiency of the energy transfer similar to that of wild type and similarly sensitive to variations in acceptor-to-donor ratios was detected (Fig. 4, A and B, and Table 1). Login to comment
179 A and B, imaging FRET of cells expressing appropriately tagged W590S or C1477R, two ABCA1 variants associated with Tangier disease and variably affecting the functional readouts associated with ABCA1 expression. Login to comment
181 C, upper panel, native PAGE profiles of lysates from cells transfected with ABCA1Y, W590S, W590SY, or C1477RY indicate that the two variants associated in dimers. Login to comment

[hide] Lipid efflux by the ATP-binding cassette transport... Biochim Biophys Acta. 2006 Jul;1761(7):655-66. Epub 2006 May 4. Cavelier C, Lorenzi I, Rohrer L, von Eckardstein A
Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1.
Biochim Biophys Acta. 2006 Jul;1761(7):655-66. Epub 2006 May 4., [PMID:16798073]

Abstract [show]
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?

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129 All ABCA1 mutants - except ABCA1(W590S) - which fail to promote cholesterol efflux also fail to cross-link apoA-I [55,56]. Login to comment
131 Interestingly, the ABCA1 (W590S) mutant cross-links apoA-I better than the wild-type ABCA1 but fails to mediate cholesterol efflux. Login to comment
136 The carboxy-terminal truncation mutant however showed reduced binding affinity to normal ABCA1 and ABCA1 (W590S). Login to comment
130 All ABCA1 mutants - except ABCA1(W590S) - which fail to promote cholesterol efflux also fail to cross-link apoA-I [55,56]. Login to comment
132 Interestingly, the ABCA1 (W590S) mutant cross-links apoA-I better than the wild-type ABCA1 but fails to mediate cholesterol efflux. Login to comment
137 The carboxy-terminal truncation mutant however showed reduced binding affinity to normal ABCA1 and ABCA1 (W590S). Login to comment

[hide] Apolipoprotein A-I activates Cdc42 signaling throu... J Lipid Res. 2006 Apr;47(4):794-803. Epub 2006 Jan 28. Nofer JR, Remaley AT, Feuerborn R, Wolinnska I, Engel T, von Eckardstein A, Assmann G
Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter.
J Lipid Res. 2006 Apr;47(4):794-803. Epub 2006 Jan 28., [PMID:16443932]

Abstract [show]
It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.

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191 These findings suggest that ABCA1-DC is able to bind apoA-I in a nearly normal manner but that this process can be uncoupled from the ABCA1-mediated outside-in signaling and the transfer of cholesterol to apoA-I. Likewise, Fitzgerald et al. (7) reported that the W590S ABCA1 mutant is fully competent to form complexes with apoA-I but fails to efflux cholesterol normally. Login to comment
189 These findings suggest that ABCA1-DC is able to bind apoA-I in a nearly normal manner but that this process can be uncoupled from the ABCA1-mediated outside-in signaling and the transfer of cholesterol to apoA-I. Likewise, Fitzgerald et al. (7) reported that the W590S ABCA1 mutant is fully competent to form complexes with apoA-I but fails to efflux cholesterol normally. Login to comment

[hide] Role of apoA-I, ABCA1, LCAT, and SR-BI in the biog... J Mol Med (Berl). 2006 Apr;84(4):276-94. Epub 2006 Feb 25. Zannis VI, Chroni A, Krieger M
Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL.
J Mol Med (Berl). 2006 Apr;84(4):276-94. Epub 2006 Feb 25., [PMID:16501936]

Abstract [show]
The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.

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147 In vitro analysis of the effects on apoA-I/ABCA1 interactions (cross-linking assay) by mutations in ABCA1 that are found in Tangier disease patients and diminish lipid efflux [71] showed that cross-linking was dramatically reduced to 5-10% of the WT control for three mutants (Gln597Arg, Cys1477Arg, and Ser1506Leu), reduced by 50% for the Arg587Trp mutant, and was remarkably increased to 125% of control for the Trp590Ser mutant [71]. Login to comment
148 Additional analysis of interactions of the mutant forms of apoA-I with the Trp590Ser mutant raised the possibility that the Trp590Ser mutation may have altered the environment in the binding site of ABCA1 in a way that prevented efficient lipid efflux despite the increased binding [73]. Login to comment
163 ApoA-I/ABCA1 binding, which exhibits a half-life of approximately 30 min [77], appears to be a necessary but not sufficient step for lipid efflux (see description of the Trp590Ser mutant above) [33, 34, 71, 80]. Login to comment
165 This appears to require the formation of a productive complex between apoA-I and ABCA1 because in the case of the ABCA1 [Trp590Ser] mutant that is associated with Tangier disease [31, 32, 71], an apoA1/ABCA1 complex is formed but the apoA-I is not lipidated and is subsequently released in a lipid-free form [77]. Login to comment
264 For instance, the binding of WT apoA-I to ABCA1[Trp590Ser] may not be productive and as a consequence, results in defective lipid efflux. Login to comment

[hide] Variations on a gene: rare and common variants in ... Annu Rev Nutr. 2006;26:105-29. Brunham LR, Singaraja RR, Hayden MR
Variations on a gene: rare and common variants in ABCA1 and their impact on HDL cholesterol levels and atherosclerosis.
Annu Rev Nutr. 2006;26:105-29., [PMID:16704350]

Abstract [show]
Cholesterol and its metabolites play a variety of essential roles in living systems. Virtually all animal cells require cholesterol, which they acquire through synthesis or uptake, but only the liver can degrade cholesterol. The ABCA1 gene product regulates the rate-controlling step in the removal of cellular cholesterol: the efflux of cellular cholesterol and phospholipids to an apolipoprotein acceptor. Mutations in ABCA1, as seen in Tangier disease, result in accumulation of cellular cholesterol, reduced plasma high-density lipoprotein cholesterol, and increased risk for coronary artery disease. To date, more than 100 coding variants have been identified in ABCA1, and these variants result in a broad spectrum of biochemical and clinical phenotypes. Here we review genetic variation in ABCA1 and its critical role in cholesterol metabolism and atherosclerosis in the general population.

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550 However, a subset of mutations are associated with greater than 50% of control HDL levels, specifically T929I, A947V, R1680W, and W590S. Login to comment
555 Since a complete loss of function allele would be expected to result in a 50% reduction in HDL levels, a greater than 50% reduction in HDL is most likely explained by a dominant negative allele, in which TABLE 3 Patient phenotypes associated with heterozygous ABCA1 mutations Mutation HDL (mmol/L) HDL (% of control) Number of patients M1091T 0.48 ± 0.5 30 ± 30 4 G1216V 0.50 40 1 R2144X 0.56 ± 0.2 41 ± 18 12 R282X 0.52 41 1 R909X 0.59 ± 0.3 42 ± 19 5 K776N 0.55 ± 0.1 47 ± 5 2 R587W 0.61 ± 0.1 47 ± 8 7 S364C 0.60 48 1 P1065S 0.80 51 1 c-ter deletion 0.75 53 1 N1800H - 56.5 33 P85L 0.72 ± 0.4 57 ± 33 5 Del693L 0.79 ± 0.2 57 ± 15 8 D1289N 0.80 ± 0.1 59 ± 12 4 R2081W 0.80 ± 0.1 59 ± 12 4 2203X 0.80 ± 0.2 59 ± 20 4 DelED1893,4 0.77 ± 0.2 59 ± 18 8 2145X 0.82 ± 0.1 59 ± 9 4 A1046D 0.70 ± 0.1 60 ± 8 2 Q597R 0.82 ± 0.1 60 ± 5 5 C1477R 0.82 ± 0.2 61 ± 15 9 IVS25 + 1G > C 0.78 ± 0.1 62 ± 12 4 D1099Y 0.83 ± 0.3 63 ± 21 5 1552X 1.00 64 1 F2009S 0.82 ± 0.2 64 ± 19 6 R587W 0.86 ± 0.1 65 ± 17 2 R1068H 0.90 ± 0.3 67 ± 26 9 N935S 1.00 ± 0.3 74 ± 16 7 T929I 1.01 ± 0.2 76 ± 7 8 1284X 1.11 ± 0.2 83 ± 14 5 A937V 1.15 ± 0.6 85 ± 28 2 R1680W 1.22 ± 0.2 87 ± 17 3 635X 1.24 ± 0.5 90 ± 32 7 W590S 1.32 ± 0.6 103 ± 46 15 the mutant protein actually interferes with the activity of the remaining wild-type protein. Login to comment

[hide] Accurate prediction of the functional significance... PLoS Genet. 2005 Dec;1(6):e83. Epub 2005 Dec 30. Brunham LR, Singaraja RR, Pape TD, Kejariwal A, Thomas PD, Hayden MR
Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene.
PLoS Genet. 2005 Dec;1(6):e83. Epub 2005 Dec 30., [PMID:16429166]

Abstract [show]
The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r2 = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation.

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48 This SNP has been reported to be associated with decreased HDL cholesterol and increased severity of atherosclerosis in Table 1. subPSEC Scores and Probability of Functional Impairment (Pdeleterious) for ABCA1 Mutations and SNPs Mutations SNPs Variant SubPSEC Pdeleterious Variant subPSEC Pdeleterious P85L À4.62 0.83 R219K À0.57 0.08 H160F À2.79 0.45 V399A À2.26 0.32 R230C À4.27 0.78 V771M À2.86 0.46 A255T À1.81 0.23 T774P À1.99 0.27 E284K À2.34 0.34 K776N À3.53 0.63 Y482C À4.21 0.77 V825I À1.06 0.13 R587W À6.04 0.95 I883M À1.38 0.17 W590S À5.19 0.9 E1172D À1.96 0.26 W590L À4.48 0.82 R1587K À0.58 0.08 Q597R À7.15 0.98 S1731C À4.21 0.77 T929I À4.29 0.78 N935H À8.54 1 N935S À7.53 0.99 A937V À6.6 0.97 A1046D À7.52 0.99 M1091T À3.56 0.64 D1099Y À6.09 0.96 D1289N À2.48 0.37 L1379F À3.81 0.69 C1477R À5.44 0.92 S1506L À5.17 0.9 N1611D À5.69 0.94 R1680W À6.02 0.95 V1704D À3.21 0.55 N1800H À4.23 0.77 R1901S À5.06 0.89 F2009S À2.73 0.43 R2081W À8.08 0.99 P2150L À2.88 0.47 Q2196H À2.74 0.43 DOI: 10.1371/journal.pgen.0010083.t001 PLoS Genetics | www.plosgenetics.org December 2005 | Volume 1 | Issue 6 | e83 0740 Accurate Prediction of ABCA1 Variants Synopsis A major goal of human genetics research is to understand how genetic variation leads to differences in the function of genes. Login to comment
75 Cholesterol Efflux Values for 293 Cells Transfected with ABCA1 Variants and subPSEC and PolyPhen Predictions of the Functional Impact of these Variants Variant Variant Type subPSEC Cholesterol Efflux PolyPhen R2081W Mutation À8.08 21.1 6 21%* Probably damaging N935S Mutation À7.53 29.3 6 13%* Benign A1046D Mutation À7.52 16.8 6 7%* Possibly damaging Q597R Mutation À7.15 17.7 6 14%* Probably damaging R587W Mutation À6.04 31.7 6 33%* Probably damaging C1477R Mutation À5.44 20.5 6 10%* Probably damaging W590S Mutation À5.19 47.1 6 13%* Probably damaging S1506L Mutation À5.17 17.8 6 15%* Probably damaging T929I Mutation À4.29 69.9 6 11%* Possibly damaging N1800H Mutation À4.23 31.3 6 16%* Possibly damaging S1731C SNP À4.21 12.3 6 10%* Possibly damaging M1091T Mutation À3.56 6.9 6 20%* Probably damaging P2150L Mutation À2.88 88.4 6 21% Probably damaging V771M SNP À2.86 145.4 6 33% Benign D1289N Mutation À2.48 137.7 6 86% Benign I883M SNP À1.38 69.1 6 16%* Benign R219K SNP À0.57 103.7 6 21.05 Benign Wild-type - 0.0 100% - *p , 0.01 compared to wild-type ABCA1. Login to comment
110 DOI: 10.1371/journal.pgen.0010083.g003 Table 3. subPSEC Scores for ABCA1 Variants Described in a Cohort of Individuals with Low HDL Cholesterol from the General Population Variant subPSEC Score Macrophage Efflux PolyPhen D1706N À6.57 0.38a Possibly damaging C1477F À5.55 0.34a Probably damaging W590S À5.19 - Probably damaging H551D À4.99 0.32a Probably damaging P85L À4.62 0.8 Probably damaging W590L À4.48 0.31a Probably damaging N1800H À4.23 0.27a Possibly damaging R965C À4.22 0.59 Probably damaging S1731C À4.21 0.28a Possibly damaging A1670T À4.2 - Possibly damaging K401Q À4.2 - Benign T459P À4.11 0.28a Possibly damaging R638Q À4.08 - Possibly damaging L1026P À3.86 0.25a Benign T2073A À3.84 0.28a Possibly damaging E815G À3.53 - Probably damaging R1615Q À3.45 - Possibly damaging S1181F À3.44 - Possibly damaging R306H À3.31 - Benign E1386Q À2.44 0.51 Benign S1376G À2.19 - Benign R1341T À2.09 - Possibly damaging D2243E À1.6 - Benign P248A À0.18 - Benign a Efflux value is 2 SDs or more below control levels of 0.52 6 0.07. Login to comment

[hide] ATP-binding cassette transporter A1: a cell choles... Physiol Rev. 2005 Oct;85(4):1343-72. Oram JF, Heinecke JW
ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease.
Physiol Rev. 2005 Oct;85(4):1343-72., [PMID:16183915]

Abstract [show]
Blood high-density lipoprotein (HDL) levels are inversely related to risk for cardiovascular disease, implying that factors associated with HDL metabolism are atheroprotective. One of these factors is ATP-binding cassette transporter A1 (ABCA1), a cell membrane protein that mediates the transport of cholesterol, phospholipids, and other metabolites from cells to lipid-depleted HDL apolipoproteins. ABCA1 transcription is highly induced by sterols, a major substrate for cellular export, and its expression and activity are regulated posttranscriptionally by diverse processes. Liver ABCA1 initiates formation of HDL particles, and macrophage ABCA1 protects arteries from developing atherosclerotic lesions. ABCA1 mutations can cause a severe HDL deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Genetic manipulations of ABCA1 expression in mice also affect plasma HDL levels and atherogenesis. Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and decrease cholesterol export from macrophages. Moreover, oxidative modifications of HDL found in patients with cardiovascular disease reduce the ability of apolipoproteins to remove cellular cholesterol by the ABCA1 pathway. These observations raise the possibility that an impaired ABCA1 pathway contributes to the enhanced atherogenesis associated with common inflammatory and metabolic disorders. The ABCA1 pathway has therefore become an important new therapeutic target for treating cardiovascular disease.

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158 One substitution mutation (W590S), however, severely reduces apolipoprotein-mediated lipid efflux without having much effect on apolipoprotein binding (73). Login to comment
414 Only one mutant (W590S) has been described that has near-normal apolipoprotein binding activity but defective lipid transport (73). Login to comment
159 One substitution mutation (W590S), however, severely reduces apolipoprotein-mediated lipid efflux without having much effect on apolipoprotein binding (73). Login to comment
413 Only one mutant (W590S) has been described that has near-normal apolipoprotein binding activity but defective lipid transport (73). Login to comment

[hide] Scavenger receptor BI and ATP-binding cassette tra... Curr Opin Lipidol. 2005 Jun;16(3):307-15. Van Eck M, Pennings M, Hoekstra M, Out R, Van Berkel TJ
Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis.
Curr Opin Lipidol. 2005 Jun;16(3):307-15., [PMID:15891392]

Abstract [show]
PURPOSE OF REVIEW: The appearance of scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in macrophages and liver implicates these transporters in different stages of reverse cholesterol transport. This review focuses on the role of SR-BI and ABCA1 in reverse cholesterol transport in the context of atherosclerotic lesion development. RECENT FINDINGS: Recent studies indicate that hepatic expression of ABCA1 and SR-BI is important for the generation of nascent HDL and the delivery of HDL cholesteryl esters to the liver, respectively. Although macrophage SR-BI and ABCA1 do not contribute significantly to circulating HDL levels, the perpetual cycle of HDL lipidation and delipidation by the liver ensures the availability of acceptors for cholesterol efflux that maintain cholesterol homeostasis in arterial macrophages, thereby reducing atherogenesis. In addition to its established role in the selective uptake of HDL cholesteryl esters, there is now evidence that hepatic SR-BI facilitates postprandial lipid metabolism, and that hepatic secretion of VLDL is dependent on ABCA1-mediated nascent HDL formation. Thus, remnant and HDL metabolism are more intimately intertwined in hepatic lipid metabolism than has previously been appreciated. SUMMARY: Recent advances in the understanding of the role of ABCA1 and SR-BI in HDL metabolism and their atheroprotective properties indicate the significant potential of modulating ABCA1 and SR-BI expression in both arterial wall macrophages and the liver for the treatment of atherosclerotic coronary artery disease.

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33 However, Fitzgerald and colleagues [32 ] recently demonstrated that a mutant form of ABCA1 (W590S), which avidly binds apoA-I but fails to promote lipid efflux to apoA-I, released apoA-I with the same kinetics as wild-type ABCA1, indicating that release of apoA-I from ABCA1 is independent of lipid transfer. Login to comment

[hide] ABC proteins: key molecules for lipid homeostasis. Med Mol Morphol. 2005 Mar;38(1):2-12. Takahashi K, Kimura Y, Nagata K, Yamamoto A, Matsuo M, Ueda K
ABC proteins: key molecules for lipid homeostasis.
Med Mol Morphol. 2005 Mar;38(1):2-12., [PMID:16158173]

Abstract [show]
Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein-cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.

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90 Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster amino acids 587 to 59746,48,72 (see Fig. 2). Login to comment
92 In contrast, the W590S mu- Fig. 2. Login to comment
95 Immunostaining with the antibody against ECD1 confirmed the proper orientation of W590S. Login to comment
96 However, the apoA-I-mediated release of cholesterol and choline phospholipids from cells expressing W590S was as low as that of the other two mutants and less than 10% of that in cells expressing the wild-type ABCA1. Login to comment
97 The W590S mutation does not have any apparent effect on ATP hydrolysis or the interaction between apoA-I and ABCA1. Login to comment
98 Analyses of W590S should facilitate our understanding of the mechanism of HDL formation. Login to comment

[hide] Screening for functional sequence variations and m... Atherosclerosis. 2004 Aug;175(2):269-79. Probst MC, Thumann H, Aslanidis C, Langmann T, Buechler C, Patsch W, Baralle FE, Dallinga-Thie GM, Geisel J, Keller C, Menys VC, Schmitz G
Screening for functional sequence variations and mutations in ABCA1.
Atherosclerosis. 2004 Aug;175(2):269-79., [PMID:15262183]

Abstract [show]
Mutations in the ATP-binding cassette 1 transporter gene (ABCA1) are responsible for the genetic HDL-deficiency syndromes, which are characterized by severely diminished plasma HDL-C levels and a predisposition to cardiovascular disease and splenomegaly. The ABCA1 gene contains 50 exons and codes for a 2261-amino acid long membrane protein that facilitates phospholipid and cholesterol transport. Several mutations have been identified so far as responsible either for Tangier disease or for reduced HDL levels. We have selectively looked for additional polymorphisms in functionally relevant regions of the gene in cohorts constituted of individuals with altered HDL levels as well as healthy blood donors and octogenarians, and screened for mutations in the complete coding region of selected individuals with extremely aberrant HDL levels. In the promoter region, which is important for regulation of gene expression, we have identified several polymorphisms including one VNTR polymorphism, located at a putative ZNF202 binding site, which displayed different binding of ZNF202 in an electromobility shift assay. Three novel SNPs were discovered in the promoter region (G1047C, C1152T and C1440T). The prevalence of exchange G1047C (G-395C) was found significantly increased in probands with low HDL compared to probands with high HDL. Exchanges C1152T (C-290T) and C1440T (C-7T) were significantly more frequent in the cohort with low HDL compared to healthy blood donors and octogenarians. In the C-terminal part of ABCA1, known to interact with other proteins, two novel sequence variations (F2163S and V2244I) have been found in one phenotype related to cardiovascular disease, but none in the aforementioned cohorts. In one individual with extremely high HDL levels, the V771M polymorphism was found in a homozygous state. In patients with HDL deficiency, three novel mutations have been identified (W590L, W840R and R1068C). To facilitate further research in ABCA1 sequence variations and expand our understanding of their effects, we are introducing a webpage archive (http://www.abca1-mutants.all.at) containing all sequence variations reported in ABCA1 so far. This webpage provides a more recent and detailed summary of sequence variations and mutations in ABCA1 than existing databases and should also be of interest for molecular diagnosis of ABCA1-related HDL deficiency.

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187 A mutation in the same codon (W590S) has already been described in a Tangier patient previously analyzed in our laboratory [7]. Login to comment
245 This is supported by the fact that a mutation in the same codon was shown to cause Tangier disease (W590S) [7], but since the patient is only heterozygous for W590L, it remains unclear what additional mutation in ABCA1 causes the patients analphalipoproteinemia. Login to comment

[hide] Alpha1-syntrophin modulates turnover of ABCA1. J Biol Chem. 2004 Apr 9;279(15):15091-5. Epub 2004 Jan 13. Munehira Y, Ohnishi T, Kawamoto S, Furuya A, Shitara K, Imamura M, Yokota T, Takeda S, Amachi T, Matsuo M, Kioka N, Ueda K
Alpha1-syntrophin modulates turnover of ABCA1.
J Biol Chem. 2004 Apr 9;279(15):15091-5. Epub 2004 Jan 13., [PMID:14722086]

Abstract [show]
ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein. Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h. To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ (PSD95-Discs large-ZO1) proteins, alpha1-syntrophin and Lin7, were found to interact with ABCA1. Immunoprecipitation revealed that alpha1-syntrophin interacted with ABCA1 strongly and that the interaction was via the C-terminal three amino acids SYV of ABCA1. Co-expression of alpha1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing alpha1-syntrophin. This effect is not common among PDZ-containing proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1. Co-expression of alpha1-syntrophin significantly increased the apoA-I-mediated release of cholesterol. ABCA1 was co-immunoprecipitated with alpha1-syntrophin from mouse brain. These results suggest that alpha1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release.

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154 A mutation of ABCA1 that causes Tangier disease (W590S) does not affect apoA-I binding or initial ATP binding/hydrolysis but results in a defect in lipid efflux (11, 31, 32). Login to comment
155 Because apoA-I failed to affect calpain degradation of ABCA1-W590S in HEK293 cells (10), additional signals following apoA-I binding to ABCA1 are speculated to be necessary for the subsequent inhibition of calpain degradation. Login to comment
156 Because apoA-I failed to affect calpain degradation of ABCA1-W590S in HEK293 cells (10), additional signals following apoA-I binding to ABCA1 are speculated to be necessary for the subsequent inhibition of calpain degradation. Login to comment

[hide] Probing the pathways of chylomicron and HDL metabo... Curr Opin Lipidol. 2004 Apr;15(2):151-66. Zannis VI, Chroni A, Kypreos KE, Kan HY, Cesar TB, Zanni EE, Kardassis D
Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer.
Curr Opin Lipidol. 2004 Apr;15(2):151-66., [PMID:15017358]

Abstract [show]
PURPOSE OF THE REVIEW: This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. RECENT FINDINGS: Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical alpha-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-beta-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. SUMMARY: New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

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228 Previous cross-linking studies of ABCA1 mutants associated with Tangier disease showed that, with the exception of one mutant, namely ABCA1 [W590S], all other ABCA1 mutants failed to cross-link or cross-linked poorly with apoA-I [59]. Login to comment
229 Mutant apoA-I forms exhibited differences in binding to the wild-type ABCA1 and W590S mutant, indicating that the W590S mutation may have altered the environment in the binding site of ABCA1 [58.. ]. Login to comment
279 Previous studies showed that an ABCA1 mutant, ABCA1[W590S], which has defective lipid efflux and causes Tangier disease [59], cross-links more efficiently to apoA-I than does wild-type ABCA1 at 378C. Login to comment
280 Analysis of the dissociation of the complexes formed between apoA-I and ABCA1 showed that the rate of dissociation was similar for the wild-type ABCA1 and the ABCA1[W590S] mutant [63. Login to comment
282 However, the apoA-I released from wild-type ABCA1 was bound to lipids, whereas the apoA-I released from ABCA1[W590S] was lipid-free [63. Login to comment
291 It has been shown that wild-type and mutant forms of apoA-I cross-link with different efficiencies to wild-type ABCA1 and the W590S mutant, suggesting different environment in the active site of the wild-type and the mutant ABCA1 [58.. ]. Login to comment
292 As a result of these differences, the wild-type apoA-I forms a complex with ABCA1[W590S] but is released lipid-free [63. Login to comment

[hide] Cross-linking and lipid efflux properties of apoA-... Biochemistry. 2004 Feb 24;43(7):2126-39. Chroni A, Liu T, Fitzgerald ML, Freeman MW, Zannis VI
Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1.
Biochemistry. 2004 Feb 24;43(7):2126-39., [PMID:14967052]

Abstract [show]
To explore the functional interactions between apoA-I and ABCA1, we correlated the cross-linking properties of several apoA-I mutants with their ability to promote cholesterol efflux. In a competitive cross-linking assay, amino-terminal deletion and double amino- and carboxy-terminal deletion mutants of apoA-I competed effectively the cross-linking of WT (125)I-apoA-I to ABCA1, while the carboxy-terminal deletion mutant apoA-I[Delta(220-243)] competed poorly. Direct cross-linking of WT apoA-I, amino-terminal, and double deletion mutants of apoA-I to ABCA1 showed similar apparent K(d) values (49-74 nM), whereas the apparent K(d) values of the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] were increased 3-fold. Analysis of several internal deletions and point mutants of apoA-I showed that apoA-I[Delta(61-78)], apoA-I[Delta(89-99)], apoA-I[Delta(136-143)], apoA-I[Delta(144-165)], apoA-I[D102A/D103A], apoA-I[E125K/E128K/K133E/E139K], apoA-I[L141R], apoA-I[R160V/H162A], and WT apoA-I had similar ABCA1-mediated lipid efflux, and all competed efficiently the cross-linking of WT (125)I-apoA-I to ABCA1. WT apoA-I and ABCA1 could be cross-linked with a 3 A cross-linker. The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. The findings are consistent with a direct association of different combinations of apoA-I helices with a complementary ABCA1 domain. Mutations that alter ABCA1/apoA-I association affect cholesterol efflux and inhibit biogenesis of HDL.

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5 The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. Login to comment
16 To further examine the structural domains of apoA-I that affect the apoA-I/ABCA1 interactions, we tested the effect of amino-terminal, carboxy-terminal, and internal deletion mutants, as well as point mutants in the central helices of apoA-I on ABCA1-mediated lipid efflux and on the ability of apoA-I to cross-link to ABCA1. Furthermore, we tested the ability of apoA-I mutants to associate with the ABCA1[W590S] mutant, which is defective in cholesterol efflux but cross-links to apoA-I (20). Login to comment
204 Effect of Amino-Terminal, Carboxy-Terminal, and Double Amino- and Carboxy-Terminal Deletions of ApoA-I on the Ability of ApoA-I to Cross-Link to the ABCA1[W590S] Mutant. Login to comment
205 To further explore the interaction of apoA-I with ABCA1, we tested the ability of various apoA-I mutants to associate with the ABCA1[W590S] mutant. Login to comment
207 For this purpose, we performed a direct cross-linking of iodinated WT and mutant forms of apoA-I (0.2 µM) to WT ABCA1 or the ABCA1[W590S] mutant in the presence or absence of 30-fold molar excess of the corresponding unlabeled apoA-I form as described in the Experimental Procedures. Login to comment
208 As shown in Figure 5A, the WT apoA-I, the amino-terminal, and the double amino- and carboxy-terminal deletion mutants of apoA-I cross-link to ABCA1[W590S] and the cross-linking is completely inhibited by 30-fold molar excess of unlabeled apoA-I. Login to comment
209 The carboxy-terminal deletion mutants apoA-I[∆(185-243)] and apoA-I[∆(220-243)] cross-link less efficiently to either WT ABCA1 or mutant ABCA1[W590S], and the cross-linking is partially competed by 30-fold molar excess of unlabeled apolipoprotein. Login to comment
210 The WT apoA-I and the apoA-I[∆(1-41)∆(185-243)] mutant have higher and equal ability, respectively, to cross-link to ABCA1[W590S] as compared to WT ABCA1. Login to comment
211 However, the amino-terminal deletion mutant apoA-I[∆(1-41)] and the carboxy-terminal deletion mutants apoA-I[∆(185-243)] and apoA-I[∆(220-243)] show 36-64% reduced cross-linking to ABCA1[W590S] (Figure 5B). Login to comment
212 The finding suggests subtle differences in the cross-linking of WT and mutant apoA-I forms to the ABCA1[W590S] mutant as compared to the cross-linking of the same apoA-I forms to the WT ABCA1. Login to comment
241 This model is supported by the observation that, with the exception of a single mutant ABCA1[W590S], all other natural mutants in the two extracellular loops of ABCA1 tested, which are defective in cholesterol efflux, cross-link inefficiently to ABCA1 (20). Login to comment
263 With FIGURE 5: Cross-linking of 125I-labeled WT and mutant forms of apoA-I to WT ABCA1 and ABCA1[W590S]. Login to comment
264 HEK293 cells transfected with plasmids carrying the WT ABCA1 and ABCA1[W590S] mutant were incubated for 1 h at 37 °C with 0.2 µM 125I-labeled WT or the indicated mutant forms of apoA-I alone or in the presence of a 30-fold molar excess of the respective unlabeled apolipoproteins. Login to comment
270 The fmol of cross-linked WT or mutant forms of apoA-I per mg of cellular protein to WT ABCA1 and ABCA1[W590S] were calculated as described in the legend of the Figure 2D,E and are graphed to panel B. Login to comment
271 Panel B shows the total cross-linking of the indicated apoA-I forms to WT ABCA1 and the ABCA1[W590S] mutant. Login to comment
274 The % ratio of the cross-linking of each 125I-labeled apoA-I form to the ABCA1[W590S] mutant to the cross-linking of the same apoA-I form to the WT ABCA1 was also calculated and is shown in the lower part of the panel B. Login to comment
316 Interactions of WT and Mutant Forms of ApoA-I with the WT ABCA1 and the ABCA1[W590S] Mutant Differ and May Account for the Inability of the ABCA1[W590S] Mutant, Which Binds Strongly to ApoA-I, To Promote Cholesterol Efflux. Login to comment
317 The present, as well as a previous study (20), showed that the ABCA1[W590S] cross-links better than WT ABCA1 to apoA-I but fails to promote cholesterol efflux in vitro and HDL formation in vivo (7, 20). Login to comment
318 However, as shown in this study, the amino- and carboxy-terminal deletion mutants cross-link less efficiently to the ABCA1[W590S] mutant, and the double deletion mutant cross-links with similar efficiency to the WT and the mutant ABCA1 forms. Login to comment
320 If the binding of apoA-I acceptors to the ABCA1 [W590S] mutant was brought about by ABCA1-dependent changes in the cell membrane (11, 12), one would expect that apoA-I mutants that bind inefficiently to the WT ABCA1 would also bind inefficiently to the ABCA1[W590S] mutant and vice versa. Login to comment
321 One possible way then to interpret the inability of the ABCA1[W590S] mutant to promote cholesterol efflux, is to assume that the complex formed is a non-productive complex that prevents the lipidation of apoA-I that is bound to ABCA1. Login to comment
323 In fact, recent experiments have confirmed this hypothesis by showing that following binding to the WT ABCA1, the apoA-I is released associated with cholesterol, whereas apoA-I bound to the ABCA1[W590S] mutant is released in lipid-free form (49). Login to comment
333 In the case of the ABCA1[W590S] mutant, the complex is formed but the FIGURE 7: Two step model of cholesterol efflux. Login to comment
339 Formation of a non-productive complex is supported by our data, which showed that WT and mutant apoA-I have differences in their cross-linking to the WT ABCA1 and the ABCA1[W590S] mutant. Login to comment

[hide] ATP-binding cassette transporter A1: regulation of... Biochem Soc Trans. 2004 Feb;32(Pt 1):124-7. Knight BL
ATP-binding cassette transporter A1: regulation of cholesterol efflux.
Biochem Soc Trans. 2004 Feb;32(Pt 1):124-7., [PMID:14748729]

Abstract [show]
The ATP-binding cassette transporter A1 (ABCA1) is involved in the regulation of cholesterol efflux from cells. Mutations in ABCA1 give rise to familial high-density lipoprotein (HDL) deficiency and Tangier disease, which is characterized by very low levels of HDL in plasma and cholesteryl ester accumulation in tonsils and other reticuloendothelial cells. The mechanism of action of ABCA1 is still unclear, but requires the transfer of phospholipid and cholesterol to apolipoprotein A1 bound by or close to the transporter. An important factor in the regulation of ABCA1 is cholesterol itself, which provides oxysterol ligands for liver X receptors that stimulate ABCA1 transcription. ABCA1-deficient mice show increased cholesterol absorption, suggesting that ABCA1 could also help to transport dietary cholesterol back out of intestinal absorptive cells into the lumen. Thus ABCA1 is intimately connected to various aspects of the regulation of whole-body cholesterol metabolism and probably plays an important role in protecting against the development of cardiovascular disease.

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65 Furthermore, a specific mutation in the first loop (W590S) reduced efflux without affecting apoA1 binding, indicating that acceptor binding is necessary, but not sufficient, for cholesterol efflux. Login to comment
64 Furthermore, a specific mutation in the first loop (W590S) reduced efflux without affecting apoA1 binding, indicating that acceptor binding is necessary, but not sufficient, for cholesterol efflux. Login to comment

[hide] ABCA1 and amphipathic apolipoproteins form high-af... J Lipid Res. 2004 Feb;45(2):287-94. Epub 2003 Nov 16. Fitzgerald ML, Morris AL, Chroni A, Mendez AJ, Zannis VI, Freeman MW
ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux.
J Lipid Res. 2004 Feb;45(2):287-94. Epub 2003 Nov 16., [PMID:14617740]

Abstract [show]
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.

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7 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. Login to comment
127 Previously, we had demonstrated that a mutant form of ABCA1, containing a missense mutation resulting in the substitution of serine for tryptophan at amino acid position 590, produced a transporter that had greater apoA-I cross-linking activity than wild-type ABCA1. Login to comment
129 This mutant (W590S) retains the temperature dependence of cross-linking we previously demonstrated for wild-type ABCA1 (Fig. 4A), suggesting that its binding interaction Fig. 2. Login to comment
141 Thus, the dissociation of apoA-I from both wild-type ABCA1 and ABCA1(W590S) was tested. Login to comment
142 Cells expressing ABCA1, the W590S mutant, or no ABCA1 (mock) were exposed to 125I-apoA-I at 37ЊC for 1 h. Login to comment
147 Fig. 4B (top panels) shows the amount of 125I-apoA-I associated with wild-type ABCA1 or the W590S mutant as assessed by the cross-linking assay. Login to comment
148 Although the W590S mutant was found to be associated with substantially more radiolabeled apoA-I at time zero, its dissociation rate from the apolipoprotein (half-life of 30.1 min) did not differ markedly from that measured for the wild-type transporter (half-life of 24.6 min) (Fig. 4B, graph). Login to comment
153 Cells expressing the W590S mutant also released the apoA-I back into the medium at a similar rate. Login to comment
154 These results, along with the measurements of the kinetics of dissociation described above, indicate that the cholesterol efflux defect in the W590S mutant cannot be accounted for by either a failure of the apoA-I/ABCA1 complex to dissociate or an ectopic release of apoA-I. Login to comment
156 The apoA-I released from the cells expressing the W590S mutant migrated at the expected molecular weight of lipid-depleted, monomeric apoA-I (Fig. 4D). Login to comment
170 IP, immunoprecipitant. wild-type ABCA1 had acquired lipid, whereas the apoA-I released from cells expressing the W590S mutant had not. Login to comment
173 Cells that were either mock-transfected or that expressed the W590S mutant had similarly low levels of [14C]cholesterol associated with apoA-I in the medium (Fig. 4D, graph). Login to comment
174 As the W590S mutant had a similar impairment in its ability to transfer phosphatidylcholine to apoA-I (data not shown), the mutant does not provide a tool for dissociating the mechanisms of efflux of these two lipids. Login to comment
176 ApoA-I associates with the ABCA1 W590S mutant in a temperature-dependent manner and is released from the mutant complex with kinetics similar to the release from wild-type ABCA1 (wtABCA1). Login to comment
177 A: ABCA1 and the W590S mutant display a similar temperature sensitivity of complex formation. Login to comment
178 Wild-type ABCA1, the W590S mutant, or empty vector (mock)-expressing cells were incubated with 125I-apoA-I at either 37ЊC or 4ЊC for 1 h. Login to comment
182 B: ApoA-I complexes between wild-type ABCA1 and the W590S mutant turn over at a similar rate. Login to comment
183 Cells expressing ABCA1 or the W590S mutant were incubated with 125I-apoA-I at 37ЊC for 1 h and then washed with warm PBS. Login to comment
186 C: ABCA1 and the W590S mutant cells release cell-associated apoA-I with similar kinetics. Login to comment
188 Graphed is the amount of 125I-apoA-I released from wild-type ABCA1 and W590S-expressing cells relative to the mock cells (duplicate samples, ϮSD) D: Only apoA-I released from wild-type ABCA1 is associated with cholesterol. Login to comment
223 In our studies, we compared the dissociation behavior of wild-type ABCA1/apoA-I complexes with those formed with the W590S ABCA1 mutant. Login to comment
225 The W590S mutant, like wild-type ABCA1, was not able to form a cross-linkable complex with apoA-I at 4ЊC. Login to comment
226 At 37ЊC, apoA-I dissociated from the W590S mutant at a rate similar to that measured using the wild-type ABCA1 (half-life of 30 versus 25 min, respectively). Login to comment
228 These results suggest that the W590S mutant binds and releases apoA-I in a normal manner but that these processes are uncoupled from the transfer of cholesterol to the released apoA-I. Login to comment
229 Our results indicate that the W590S mutant has a similar impairment in the transfer of phospholipid, and as reported by Rigot et al. (9), the mutant does not stimulate the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. Login to comment
231 If this mechanism were operative, it seems unlikely that the apoA-I would dissociate from the W590S mutant with the kinetics we have measured. Login to comment
232 The behavior of the W590S mutant/apoA-I complex indicates that the cellular release of apoA-I and the transfer of cholesterol and phospholipid are separable events and thus likely distinct steps in the efflux mechanism. Login to comment
242 The W590S ABCA1 mutant demonstrates that this release is not dependent on cholesterol transfer to the acceptor apolipoprotein, indicating that release and cholesterol transfer are separable events in the efflux process. Login to comment
6 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. Login to comment
126 Previously, we had demonstrated that a mutant form of ABCA1, containing a missense mutation resulting in the substitution of serine for tryptophan at amino acid position 590, produced a transporter that had greater apoA-I cross-linking activity than wild-type ABCA1. Login to comment
128 This mutant (W590S) retains the temperature dependence of cross-linking we previously demonstrated for wild-type ABCA1 (Fig. 4A), suggesting that its binding interaction Fig. 2. Login to comment
140 Thus, the dissociation of apoA-I from both wild-type ABCA1 and ABCA1(W590S) was tested. Login to comment
146 Fig. 4B (top panels) shows the amount of 125I-apoA-I associated with wild-type ABCA1 or the W590S mutant as assessed by the cross-linking assay. Login to comment
152 Cells expressing the W590S mutant also released the apoA-I back into the medium at a similar rate. Login to comment
155 The apoA-I released from the cells expressing the W590S mutant migrated at the expected molecular weight of lipid-depleted, monomeric apoA-I (Fig. 4D). Login to comment
169 IP, immunoprecipitant. wild-type ABCA1 had acquired lipid, whereas the apoA-I released from cells expressing the W590S mutant had not. Login to comment
172 Cells that were either mock-transfected or that expressed the W590S mutant had similarly low levels of [14C]cholesterol associated with apoA-I in the medium (Fig. 4D, graph). Login to comment
175 ApoA-I associates with the ABCA1 W590S mutant in a temperature-dependent manner and is released from the mutant complex with kinetics similar to the release from wild-type ABCA1 (wtABCA1). Login to comment
181 B: ApoA-I complexes between wild-type ABCA1 and the W590S mutant turn over at a similar rate. Login to comment
185 C: ABCA1 and the W590S mutant cells release cell-associated apoA-I with similar kinetics. Login to comment
187 Graphed is the amount of 125I-apoA-I released from wild-type ABCA1 and W590S-expressing cells relative to the mock cells (duplicate samples, SD) D: Only apoA-I released from wild-type ABCA1 is associated with cholesterol. Login to comment
222 In our studies, we compared the dissociation behavior of wild-type ABCA1/apoA-I complexes with those formed with the W590S ABCA1 mutant. Login to comment
224 The W590S mutant, like wild-type ABCA1, was not able to form a cross-linkable complex with apoA-I at 4C. Login to comment
227 These results suggest that the W590S mutant binds and releases apoA-I in a normal manner but that these processes are uncoupled from the transfer of cholesterol to the released apoA-I. Login to comment
230 If this mechanism were operative, it seems unlikely that the apoA-I would dissociate from the W590S mutant with the kinetics we have measured. Login to comment
241 The W590S ABCA1 mutant demonstrates that this release is not dependent on cholesterol transfer to the acceptor apolipoprotein, indicating that release and cholesterol transfer are separable events in the efflux process. Login to comment

[hide] Posttranscriptional regulation of human ABCA7 and ... Biochem Biophys Res Commun. 2003 Nov 14;311(2):313-8. Ikeda Y, Abe-Dohmae S, Munehira Y, Aoki R, Kawamoto S, Furuya A, Shitara K, Amachi T, Kioka N, Matsuo M, Yokoyama S, Ueda K
Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release.
Biochem Biophys Res Commun. 2003 Nov 14;311(2):313-8., [PMID:14592415]

Abstract [show]
ABCA7 is expressed predominantly in myelo-lymphatic tissues or reticuloendothelial cells. Physiological role and function of this protein are not fully understood. We isolated the full-length cDNA (type I) and a splicing variant cDNA (type II) of human ABCA7, and developed monoclonal antibodies against extracellular domain (ECD)1 of ABCA7. RT-PCR experiments suggested that human ABCA7 gene produced the type II mRNA in a tissue-specific manner. Immunostaining revealed that the type I ABCA7, expressed in HEK293 cells, was localized to the plasma membrane and ECD1 was exposed to the extracellular space as was the case for ABCA1. HEK293 cells expressing type I ABCA7 showed apoA-I-dependent cholesterol and phospholipid release. In contrast, type II ABCA7 appeared to be localized mainly in endoplasmic reticulum and did not show apoA-I-dependent cholesterol and phospholipid release. Alternative splicing could be involved in the post-transcriptional regulation of the expression and function of human ABCA7.

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15 In fact, the three different mutants in ECD1 associated with TD, R587W, W590S, and Q597R, all had reduced apoA-I-mediated lipid release and subsequent HDL assembly when expressed in HEK293 [12-14]. Login to comment

[hide] Phosphorylation of a pest sequence in ABCA1 promot... J Biol Chem. 2003 Sep 26;278(39):37368-74. Epub 2003 Jul 17. Martinez LO, Agerholm-Larsen B, Wang N, Chen W, Tall AR
Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I.
J Biol Chem. 2003 Sep 26;278(39):37368-74. Epub 2003 Jul 17., [PMID:12869555]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the apoAI-dependent efflux of excess cholesterol from cells. We recently showed that ABCA1 proteolysis by calpain was dependent on a PEST sequence in the cytoplasmic region of ABCA1 and was reversed by apoA-I interaction with ABCA1. We show here that phosphorylation of ABCA1 in HEK293 cells was reduced by 63 +/- 2.4% after removal of the PEST sequence (ABCA1delPEST) or by incubation of cells with apoAI (58 +/- 3.3%). By contrast, ABCA1delPEST showed no further decrease of phosphorylation upon apoAI treatment. To assess the hypothesis that PEST sequence phosphorylation could regulate ABCA1 calpain proteolysis, we mutagenized S/T residues in the PEST sequence and identified Thr-1286 and Thr-1305 as constitutively phosphorylated residues. The ABCA1-T1286A/T1305A mutant was not degraded by calpain and was not further stabilized upon apoA-I treatment. The T1286A/T1305A mutant showed a 3.1-fold increase in cell surface expression and a 2.3-fold increase of apoAI-mediated cholesterol efflux compared with wild type ABCA1. In conclusion, we propose a mechanism of regulation of ABCA1 cell surface expression and function in which the interaction with apoA-I results in dephosphorylation of the ABCA1 PEST sequence and thereby inhibits calpain degradation leading to an increase of ABCA1 cell surface expression.

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165 A mutant (ABCA1-W590S) showed normal levels of binding of apoA-I but decreased phospholipid efflux (22, 23). Login to comment
162 A mutant (ABCA1-W590S) showed normal levels of binding of apoA-I but decreased phospholipid efflux (22, 23). Login to comment

[hide] Efflux and atherosclerosis: the clinical and bioch... Arterioscler Thromb Vasc Biol. 2003 Aug 1;23(8):1322-32. Epub 2003 May 22. Singaraja RR, Brunham LR, Visscher H, Kastelein JJ, Hayden MR
Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene.
Arterioscler Thromb Vasc Biol. 2003 Aug 1;23(8):1322-32. Epub 2003 May 22., [PMID:12763760]

Abstract [show]
Approximately 50 mutations and many single nucleotide polymorphisms have been described in the ABCA1 gene, with mutations leading to Tangier disease and familial hypoalphalipoproteinemia. Homozygotes and heterozygotes for mutations in ABCA1 display a wide range of phenotypes. Identification of ABCA1 as the molecular defect in these diseases has allowed for ascertainment based on genetic status and determination of genotype-phenotype correlations and has permitted us to identify mutations conferring a range of severity of cellular, biochemical, and clinical phenotypes. In this study we review how genetic variation at the ABCA1 locus affects its role in the maintenance of lipid homeostasis and the natural progression of atherosclerosis.

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76 Additional insights into how the mutations R587W, W590S, and Q597R that occur in the extracellular loops affect ABCA1 function have recently been described.58-60 Two studies have reported that ABCA1 containing the point mutation Q597R, which occurs in the first extracellular loop, does not localize to the plasma membrane.59,60 However, other studies have reported that this mutant is expressed at the plasma membrane but at reduced levels relative to wild-type ABCA1.58,44 R587W, another missense mutation in the first extracellular loop, also prevents the trafficking of ABCA1 to the plasma membrane, although results with this mutant have been variable.58-60 Both the R587W and Q597R mutants are resistant to PNGase digestion, indicating that they are not glycosylated, suggesting that ABCA1 harboring these mutations does not traverse the medial and trans Golgi network. Login to comment
78 ABCA1 harboring the W590S mutation does reach the cell surface, and cross-linking studies reported normal interaction of the W590S mutant with ApoA-I despite defective efflux, suggesting that interaction with ApoA-I may not be sufficient for lipid efflux.58 ABCA1 that does not reach the plasma membrane cannot induce the binding of ApoA-I. Login to comment
83 TABLE 2. Conservation of Amino Acid Residues Mutated in Humans Mutation H. sapiens M. musculus G. gallus D. melanogaster C. elegans P85L P P P ⅐ ⅐ ⅐ P R230C R R R P G A255T A A S ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ R587W R R R ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ W590S W W W R Q Q597R Q Q Q Q Q ⌬L693 L L L L L T929I T T T T T N935S/H N N N N N A937V A A A A A A1046D A A A A A M1091T M M M M M D1099Y D D D D D D1289L/N D D D D D C1477R C C C ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ S1506L S S S ⅐ ⅐ ⅐ ⅐ ⅐ ⅐ N1611D N N N N S R1680W R R R R R N1800H N N N A W F2009S F F F I M R2081W R R R R R P2150L P P P R N ⌬E1893 E E E D S ⌬D1894 D D D D D Twenty-three of 24 (95.83%) amino acids affected by mutations are conserved with G. gallus, reflecting the functional importance of these residues. Login to comment
114 Patients homozygous for the mutations A255T and R1680W show HDL-C levels that are greater than 10% of age-and sex-matched population controls. Login to comment
122 This is indeed the case in heterozygous patients harboring mutations A255T, W590S, T929I, R1680W, and A937V, who all show HDL-C levelsϾ75% of normal age-and sex-matched controls. Login to comment
68 Additional insights into how the mutations R587W, W590S, and Q597R that occur in the extracellular loops affect ABCA1 function have recently been described.58-60 Two studies have reported that ABCA1 containing the point mutation Q597R, which occurs in the first extracellular loop, does not localize to the plasma membrane.59,60 However, other studies have reported that this mutant is expressed at the plasma membrane but at reduced levels relative to wild-type ABCA1.58,44 R587W, another missense mutation in the first extracellular loop, also prevents the trafficking of ABCA1 to the plasma membrane, although results with this mutant have been variable.58-60 Both the R587W and Q597R mutants are resistant to PNGase digestion, indicating that they are not glycosylated, suggesting that ABCA1 harboring these mutations does not traverse the medial and trans Golgi network. Login to comment
70 ABCA1 harboring the W590S mutation does reach the cell surface, and cross-linking studies reported normal interaction of the W590S mutant with ApoA-I despite defective efflux, suggesting that interaction with ApoA-I may not be sufficient for lipid efflux.58 ABCA1 that does not reach the plasma membrane cannot induce the binding of ApoA-I. Login to comment
75 TABLE 2. Conservation of Amino Acid Residues Mutated in Humans Mutation H. sapiens M. musculus G. gallus D. melanogaster C. elegans P85L P P P ዼ ዼ ዼ P R230C R R R P G A255T A A S ዼ ዼ ዼ ዼ ዼ ዼ R587W R R R ዼ ዼ ዼ ዼ ዼ ዼ W590S W W W R Q Q597R Q Q Q Q Q èc;L693 L L L L L T929I T T T T T N935S/H N N N N N A937V A A A A A A1046D A A A A A M1091T M M M M M D1099Y D D D D D D1289L/N D D D D D C1477R C C C ዼ ዼ ዼ ዼ ዼ ዼ S1506L S S S ዼ ዼ ዼ ዼ ዼ ዼ N1611D N N N N S R1680W R R R R R N1800H N N N A W F2009S F F F I M R2081W R R R R R P2150L P P P R N èc;E1893 E E E D S èc;D1894 D D D D D Twenty-three of 24 (95.83%) amino acids affected by mutations are conserved with G. gallus, reflecting the functional importance of these residues. Login to comment

[hide] Regulation and mechanisms of ATP-binding cassette ... Arterioscler Thromb Vasc Biol. 2003 Jul 1;23(7):1178-84. Epub 2003 May 8. Wang N, Tall AR
Regulation and mechanisms of ATP-binding cassette transporter A1-mediated cellular cholesterol efflux.
Arterioscler Thromb Vasc Biol. 2003 Jul 1;23(7):1178-84. Epub 2003 May 8., [PMID:12738681]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1) plays a major role in cholesterol homeostasis and HDL metabolism. ABCA1 mediates cellular cholesterol and phospholipid efflux to lipid-poor apolipoproteins, and upregulation of ABCA1 activity is antiatherogenic. ApoA-I, the major apolipoprotein component of HDL, promotes ABCA1-mediated cholesterol and phospholipid efflux, probably by directly binding to ABCA1. ABCA1 gene expression is markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 protein turnover is rapid. ABCA1 contains a PEST--proline (P), glutamate (E), serine (S), and threonine (T)--sequence in the intracellular segment that mediates ABCA1 degradation by a thiol protease, calpain. ApoA-I and apoE stabilize ABCA1 in a novel mode of regulation by decreasing PEST sequence-mediated calpain proteolysis. ABCA1-mediated cholesterol and phospholipid efflux are distinctly regulated and affected by the activity of other gene products. Stearyol CoA desaturase decreases ABCA1-mediated cholesterol efflux but not phospholipid efflux, likely by decreasing the cholesterol pool available to ABCA1. This and other evidence suggest that ABCA1 promotes cholesterol and phospholipid efflux, probably by directly transporting both lipids as substrates.

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46 Importantly, many ABCA1 missense mutations causing Tangier disease have been identified in these loops.29 A functional test of these mutations in transfected cells revealed defects in apoA-I binding and cellular lipid efflux.27,28 Interestingly, one of the mutants, W590S, showed lipid efflux deficiency but moder- Figure 1. Login to comment
51 Together, these results also suggest that apoA-I binding requires an ABCA1 molecule assuming an optimal structural conformation that is maintained by a functional ATPase because cell-surface ABCA1 mutants with defective ATPase fail to bind apoA-I.23,32 The moderate increase in apoA-I binding to W590S may imply that dissociation of apoA-I from ABCA1 could be facilitated by lipid efflux to apolipoproteins bound to ABCA1 and is consistent with the earlier finding that ABCA1 binds apoA-I but not HDL3.7 Extrahepatic ABCA1 functions to promote cholesterol efflux from peripheral tissues to lipid-poor apolipoproteins that in turn deliver the lipid load back to liver for disposal, a process likely involving scavenger receptor BI, which has high affinity for lipid-rich HDLs but low affinity for lipid-poor apolipoproteins.33 Hepatic ABCA1 is likely involved in pre-beta HDL formation in liver, demonstrated by increased pre-beta HDL as well as more mature HDL levels in mice with adenovirus-mediated expression of ABCA1.34 Like scavenger receptor BI, ABCA1 binds not only apoA-I but also other apolipoproteins, including apoE.35 Thus, the antiatherogenic effect of apoE in vivo could be partially explained by lipid efflux from macrophage foam cells coordinated with ABCA1. Login to comment
78 It may also cause conformational changes of ABCA1, as reported for ligand-induced conformational changes of other ABC transporters.48 Cholesterol efflux appears to have no effect on ABCA1 protein levels because HDL and cyclodextrin treatment promoted cholesterol efflux to an extent similar to or greater than that by apoA-I-mediated cholesterol efflux but failed to alter ABCA1 protein levels.31 Another mutation of ABCA1 that causes Tangier disease (W590S) has been shown to cause a moderate increase in apoA-I binding but defective lipid efflux.27 ApoA-I failed to increase ABCA1-W590S levels whereas calpeptin significantly increased ABCA1-W590S proteins, suggesting that apoA-I binding is not sufficient for ABCA1 stabilization.31 These results favor the hypothesis that apoA-I-mediated ABCA1 stabilization may result from a local change in membrane phospholipids that decreases the binding of a hydrophobic, glycine-rich sequence of the small calpain subunit.49 However, an equally valid interpretation is that apoA-I fails to bind the W590S mutant in the correct orientation and therefore the appropriate conformational change of ABCA1 required to decrease calpain proteolysis does not occur. Login to comment
41 Importantly, many ABCA1 missense mutations causing Tangier disease have been identified in these loops.29 A functional test of these mutations in transfected cells revealed defects in apoA-I binding and cellular lipid efflux.27,28 Interestingly, one of the mutants, W590S, showed lipid efflux deficiency but moder- Figure 1. Login to comment
73 It may also cause conformational changes of ABCA1, as reported for ligand-induced conformational changes of other ABC transporters.48 Cholesterol efflux appears to have no effect on ABCA1 protein levels because HDL and cyclodextrin treatment promoted cholesterol efflux to an extent similar to or greater than that by apoA-I-mediated cholesterol efflux but failed to alter ABCA1 protein levels.31 Another mutation of ABCA1 that causes Tangier disease (W590S) has been shown to cause a moderate increase in apoA-I binding but defective lipid efflux.27 ApoA-I failed to increase ABCA1-W590S levels whereas calpeptin significantly increased ABCA1-W590S proteins, suggesting that apoA-I binding is not sufficient for ABCA1 stabilization.31 These results favor the hypothesis that apoA-I-mediated ABCA1 stabilization may result from a local change in membrane phospholipids that decreases the binding of a hydrophobic, glycine-rich sequence of the small calpain subunit.49 However, an equally valid interpretation is that apoA-I fails to bind the W590S mutant in the correct orientation and therefore the appropriate conformational change of ABCA1 required to decrease calpain proteolysis does not occur. Login to comment

[hide] Genetics of HDL regulation in humans. Curr Opin Lipidol. 2003 Jun;14(3):273-9. Miller M, Rhyne J, Hamlette S, Birnbaum J, Rodriguez A
Genetics of HDL regulation in humans.
Curr Opin Lipidol. 2003 Jun;14(3):273-9., [PMID:12840658]

Abstract [show]
PURPOSE OF REVIEW: To review gene regulation of HDL-cholesterol and discuss molecular abnormalities in HDL candidate genes that may lead to human pathologic states. RECENT FINDINGS: The inverse association between HDL-cholesterol and vascular disease, especially coronary heart disease, has long been recognized, but understanding gene regulation of HDL in humans gained considerable momentum following the identification of ABCA1 as playing a pivotal role in reverse cholesterol transport. Recent data suggest that potentially important targets for upregulating HDL in humans include upregulators of ABCA1 and APOA1 (e.g. peroxisome proliferator activated receptor and liver X receptor agonists) and downregulators of CETP (e.g. JTT-705). A host of other nuclear receptors under investigation in animal models may advance to human testing in the near future. SUMMARY: Disorders affecting HDL metabolism are complex because monogenic disorders causing low HDL do not necessarily correlate with premature vascular disease. To date, pathologic phenotypes have only been deduced among several HDL candidate genes. Understanding the genetic underpinnings associated with variant HDL and reverse cholesterol transport provides an exceptional opportunity to identify novel agents that may optimize this process and reduce vascular event rates beyond currently available LDL lowering therapies.

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66 TD 1591 T/C 11 V399A extracellular [68] TD 1979 (110bpAlu Ins) 12 truncated truncation [60] TD/FHA 2154 C/T 14 R587W extracellular [67,69] TD 2164 G/C 14 W590S extracellular [61] TD 2185 A/G 14 Q597R extracellular [59,67] TD 2219 G/del 14 truncated, 635X truncated [60,61] FHA 2472-2474 3bp del 15 Del L693 TM domain #3 [59] phosphorylation 2706 G/A 16 V771M extracellular [68] 2715 A/C 16 T774P extracellular [68] 2723 G/C 16 K776N extracellular [68] 2868 G/A 17 V825I TM domain #6 [67,68] TD/FHA 3044 A/G 18 I883M cytoplasmic [68] phosphorylat site FHA 3120 C/T 19 R909X truncation [63,71] TD 3181 C/T 19 T929I cytoplasmic [62] TD 3199 A/G 19 N935S Walker A [61] TD 3205 C/T 19 A937V Walker A [61] TD 3532 C/A 22 A1046D cytoplasmic, Walker A/B [70] FHA 3667 T/C 23 M1091T cytoplasmic [63] 3690 G/T 23 D1099Y cytoplasmic [9] TD 3738 2bp del 23 1145X truncation [66] FHA 3911 G/C 24 E1172D linker/cytoplasmic [68] FHA 4242 4bp del 27 1297X truncated [64] TD 4260 G/A 27 D1289N linker cytoplasm [64,65] TD 4824 T/C 31 C1477R extracellular [59] TD 4912 C/T 32 S1506L extracellular loop #2 [71] TD 5025 ins A 34 A1544S?1552X truncation [70] 5059 T/C 34 I1555T extracellular loop #2 [67] 5155 G/A 35 R1587K extracellular loop #2 [68] FHA 5226 A/G 36 N1611D extracellular loop #2 [75..] 5338 T/C 36 L1648P extracellular loop #2 [67] TD 5443 C/T 37 R1680W cytoplasmic [74.] Login to comment

[hide] Effects of mutations of ABCA1 in the first extrace... J Biol Chem. 2003 Mar 7;278(10):8815-9. Epub 2002 Dec 31. Tanaka AR, Abe-Dohmae S, Ohnishi T, Aoki R, Morinaga G, Okuhira K, Ikeda Y, Kano F, Matsuo M, Kioka N, Amachi T, Murata M, Yokoyama S, Ueda K
Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.
J Biol Chem. 2003 Mar 7;278(10):8815-9. Epub 2002 Dec 31., [PMID:12509412]

Abstract [show]
ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.

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1 The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. Login to comment
5 R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Login to comment
6 Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Login to comment
7 Photoaffinity labeling of W590S with 8-azido-[␣- 32 P]ATP was stimulated by adding ortho-vanadate in the presence of Mn2؉ as much as in the presence of wild-type ABCA1. Login to comment
8 These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis. Login to comment
20 Three TD mutants (R587W, W590S, Q597R), clustered in ECD1, were examined in the present report. Login to comment
24 On the other hand, the two mutants R587W and Q597R were only partially or scarcely localized to the PM, whereas W590S * This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas "ABC Proteins" from the Ministry of Education, Science, Sports, and Culture of Japan and by the Nakajima Foundation. Login to comment
35 Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Login to comment
44 DNA Construction-DNA fragments (XhoI-BclI) containing each missense TD mutation (R587W, W590S, or Q597R) were generated using the polymerase chain reaction method with R587W (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACATGTGGTACGTC-3Ј), W590S (XhoI) primer (5Ј-GTCCTCGAGCTGACCCCTTTGAGGACAT- GCGGTACGTCTCGGGGGGCTTC-3Ј), or Q597 (XhoI) primer (5Ј-GT- CCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGG- CTTCGCCTACTTGCGGGATGTGGTG-3Ј), where the mutated nucleotide is underlined, and BclI primer (5Ј-CGATGCCCTTGATGATCACA- GCCACTGAG-3Ј). Login to comment
47 In brief, 10 ␮g of membrane proteins from HEK293 cells stably expressing the wild-type, R587W, W590S, or Q597R ABCA1-GFP were treated with 500 units of Endo H or 0.3 units of PNGaseF for 1 h at 37 °C. Login to comment
53 Vanadate-induced Nucleotide Trapping in ABCA1 with 8-Azido-[␣- 32 P]ATP-A membrane fraction (20-30 ␮g) was prepared from HEK293 cells stably expressing the wild-type or W590S ABCA1-GFP. Login to comment
64 Effects of ECD1 Mutations on Subcellular Localization of ABCA1-GFP-Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster in the vicinity between amino acids 587 and 597 (20)(Fig. 2A). Login to comment
68 Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). Login to comment
69 In contrast, W590S was localized to the PM as much as the wild-type ABCA1-GFP was, although more was found with intracellular vesicles than with the wild type (Fig. 2B). Login to comment
70 Immunostaining with the antibody against ECD1 confirmed the proper orientation of W590S (Fig. 2C). Login to comment
71 Glycosylation of ABCA1-GFP-Glycosylation of the wild-type ABCA1-GFP and its mutants R587W, W590S, and Q597R was examined by the treatment with PNGaseF and Endo H (Fig. 3A). Login to comment
75 These results indicated that R587W and Q597R ABCA1-GFP did not contain complex oligosaccharides and supported the confocal microscopy observation, which suggested the localization of these two TD mutants in the ER or the cis-Golgi complex. Login to comment
76 On the other hand, W590S ABCA1-GFP was resistant to Endo H, indicating that it does not contain high mannose oligosaccharides but contains complex oligosaccharides and reached the trans-Golgi complex. Login to comment
87 The apoA-I-mediated release of cholesterol and choline-phospholipids from HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was localized to the PM as efficiently as the wild type. Login to comment
89 The results were similar to those observed with the stable transformants shown in Fig. 4, A and B. Interaction of ABCA1-GFP with 8-Azido-[␣-32 P]ATP-To elucidate the mechanism for the loss of function of ABCA1 in the W590S mutant, we examined the interaction of ABCA1-GFP with ATP. Login to comment
102 A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment
103 B, GFP fluorescence of HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP and three TD mutants R587W, W590S, and Q597R ABCA1-GFP. Login to comment
104 C, immunofluorescent observation of W590S ABCA1-GFP with anti-ECD1 antibody and anti-rat IgG-Alexa594. Login to comment
107 The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (-), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment
111 Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 ␮g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 ␮g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment
113 To determine whether the W590S mutation affects the ATP hydrolysis cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP was examined in the presence of Mn2ϩ (Fig. 5C). Login to comment
114 Membrane proteins from HEK293 cells expressing a similar amount of wild-type ABCA1 or W590S ABCA1-GFP (Fig. 5B) were incubated with 8-azido-[␣-32 P]ATP in the absence or presence of ortho-vanadate. Login to comment
115 The photoaffinity labeling of W590S ABCA1-GFP was stimulated by adding ortho-vanadate in the presence of Mn2ϩ as much as in the presence of the wild type. Login to comment
119 ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment
120 On the other hand, W590S ABCA1-GFP was mainly localized to the PM as much as the wild-type ABCA1 was. Login to comment
122 R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. Login to comment
123 In contrast, W590S ABCA1-GFP contained complex-type oligosaccharides as the wild-type does. Login to comment
127 This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Login to comment
130 W590S ABCA1-GFP was localized to the PM as much as the wild-type ABCA1-GFP when expressed in HEK293. Login to comment
132 To elucidate the reason for this functional impairment in the W590S mutant, nucleotide interaction was examined with the wild-type and W590S ABCA1-GFP by using 8-azido-[␣-32 P]ATP. Login to comment
139 B, immunoblots of membranes prepared from HEK293 cells stably expressing the wild-type (4 ␮g, lane 1) or W590S (6 ␮g, lane 2) ABCA1-GFP or from untransfected HEK293 cells (6 ␮g, lane 3). Login to comment
141 C, membranes prepared from HEK293 cells stably expressing the wild type (WT) (20 ␮g, lanes 1 and 2) or W590S (30 ␮g, lanes 3 and 4) were incubated with 15 ␮M 8-azido-[␣-32 P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 °C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures." Login to comment
146 W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the presence of Mn2ϩ . Login to comment
148 It has been reported that apoA-I does not properly interact with ATP hydrolysis mutants of ABCA1 (10) and that apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1 (17, 35). Login to comment
149 These results suggest that W590S ABCA1-GFP possesses, at least, minimum ATPase activity, which supports apoA-I binding. Login to comment
150 W590S mutation may impair a step after the interaction with apoA-I, such as proper loading of phospholipid and/or cholesterol or proper release of apoA-I after phospholipid/cholesterol loading. Login to comment
154 Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment
155 In this study, we demonstrated that the defect of HDL assembly in R587W and Q597R is due to the impaired localization of ABCA1 to the PM. Login to comment
156 Subcellular trafficking and vanadate-induced nucleotide trapping in the presence of Mn2ϩ were not impaired in ABCA1-GFP containing the W590S mutation so that W590S seems to have a different type of functional defect. Login to comment
86 The apoA-I-mediated release of cholesterol and choline-phospholipids from HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was localized to the PM as efficiently as the wild type. Login to comment
88 The results were similar to those observed with the stable transformants shown in Fig. 4, A and B. Interaction of ABCA1-GFP with 8-Azido-[ॷ-32 P]ATP-To elucidate the mechanism for the loss of function of ABCA1 in the W590S mutant, we examined the interaction of ABCA1-GFP with ATP. Login to comment
101 A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. Login to comment
106 The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (afa;), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Login to comment
110 Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 òe;g/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 òe;g/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Login to comment
112 To determine whether the W590S mutation affects the ATP hydrolysis cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP was examined in the presence of Mn2af9; (Fig. 5C). Login to comment
126 This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Login to comment
129 W590S ABCA1-GFP was localized to the PM as much as the wild-type ABCA1-GFP when expressed in HEK293. Login to comment
131 To elucidate the reason for this functional impairment in the W590S mutant, nucleotide interaction was examined with the wild-type and W590S ABCA1-GFP by using 8-azido-[ॷ-32 P]ATP. Login to comment
138 B, immunoblots of membranes prepared from HEK293 cells stably expressing the wild-type (4 òe;g, lane 1) or W590S (6 òe;g, lane 2) ABCA1-GFP or from untransfected HEK293 cells (6 òe;g, lane 3). Login to comment
140 C, membranes prepared from HEK293 cells stably expressing the wild type (WT) (20 òe;g, lanes 1 and 2) or W590S (30 òe;g, lanes 3 and 4) were incubated with 15 òe;M 8-azido-[ॷ-32 P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 &#b0;C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures." Login to comment
145 W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the presence of Mn2af9; . Login to comment
147 It has been reported that apoA-I does not properly interact with ATP hydrolysis mutants of ABCA1 (10) and that apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1 (17, 35). Login to comment
153 Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. Login to comment

[hide] Distinct sites on ABCA1 control distinct steps req... J Lipid Res. 2002 Dec;43(12):2077-86. Rigot V, Hamon Y, Chambenoit O, Alibert M, Duverger N, Chimini G
Distinct sites on ABCA1 control distinct steps required for cellular release of phospholipids.
J Lipid Res. 2002 Dec;43(12):2077-86., [PMID:12454269]

Abstract [show]
The loss of ABCA1 function leads to Tangier dyslipidemia in humans and to a Tangier-like phenotype in mice, by impairing the transformation of nascent apolipoproteins into mature HDL particles. Mechanistically this ensues from the inability of cells to release membrane lipids and cholesterol. Whereas the ability of ABCA1 to promote phospholipid effluxes, surface binding of apolipoproteins and outward flip of membrane lipids has been documented, the relationship between this series of ABCA1-dependent events is still elusive. Here we provide evidence that i) lipid effluxes require both flip of membrane lipids and binding of apolipoproteins to the cell surface, ii) apolipoprotein A-I binding depends on structural determinants on ABCA1, and iii) phospholipid effluxes can be modulated by engineered mutations on the structural determinants identified on ABCA1.

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140 Three point mutations in the region 580-600 had been reported in Tangier pedigrees (namely R587W, W590S, and Q597R). Login to comment
148 This was virtually complete in the case of W590S, Q597R, and ⌬L693, and reduced to one fourth for R587W and C1477R (Table 3). Login to comment
170 The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment
171 Indeed, these variants, while eliciting an apoA-I binding indistinguishable from wild-type ABCA1 (79% Ϯ 5, n ϭ 7, P Ͼ 0.05 and 126% Ϯ 18, n ϭ 6, P Ͼ 0.05 of wild type, respectively) fail to drive both flipping of PS (annexin V binding ϭ 39% Ϯ 11of wild type for R587W, n ϭ 5, P Ͻ 0.05 and 30% Ϯ 9 for W590S, n ϭ 7, P Ͻ 0.01) and membrane release of PL (27% Ϯ 16, n ϭ 3, P Ͻ 0.05 and 16% Ϯ 3, n ϭ 2, P Ͻ 0.01 of wild type, respectively, Table 3), thus indicating that apoA-I binding per se is insufficient for the generation of PL effluxes, which also requires PS flipping. Login to comment
205 Morphological and functional evaluation of Tangier- associated ABCA1 variants Identity SL AnnV St ApoA-I St PL Efflux St % % % R587W PM 39 Ϯ 11(5) a 79 Ϯ 5 (7) a 27 Ϯ 16 (3) a W590S PM 30 Ϯ 9 (7) b 126 Ϯ 18 (6) ns 16 Ϯ 3 (2) b Q597R ER, PM 24 Ϯ 9 (4) b 15 Ϯ 8 (4) c 8 Ϯ 7 (2) b ⌬L693 ER 26 Ϯ 11 (5) a 12 Ϯ 6 (4) c nd C1477R PM 53 Ϯ 12 (9) ns 33 Ϯ 9 (6) b 12 Ϯ 2 (2) b HA819/1466 PM 54 Ϯ 12 (6) ns 49 Ϯ 7 (6) a 108 Ϯ 28 (4) b HA819/C1477R PM 57 Ϯ 15 (4) ns 68 Ϯ 6 (4) a 142 Ϯ 24 (2) b SL, subcellular localization as detected by confocal imaging and confirmed by surface biotynilation; AnnV and ApoA-I, binding of annexin V or apoA-I in cells successfully transfected with the test construct (GFP positive); St, statistical significance. Login to comment
217 This is the case of the Tangier variant W590S, whose behavior has also been documented by Fitzgerald et al. (22). Login to comment
139 We deliberately excluded mutations in the nucleotide binding folds, i.e., those expected to impair function by interference with the ATPase activity, and conversely selected the mutations located in the extracellular region defined by the topological model proposed in Fig. 4A. Three point mutations in the region 580-600 had been reported in Tangier pedigrees (namely R587W, W590S, and Q597R). Login to comment
147 This was virtually complete in the case of W590S, Q597R, and L693, and reduced to one fourth for R587W and C1477R (Table 3). Login to comment
169 The last category is illustrated by R587W and W590S Tangier transporters, which are correctly targeted to the plasma membrane, but show a marked functional dissociation. Login to comment
204 Morphological and functional evaluation of Tangier-associated ABCA1 variants Identity SL AnnV St ApoA-I St PL Efflux St % % % R587W PM 39 11(5) a 79 5 (7) a 27 16 (3) a W590S PM 30 9 (7) b 126 18 (6) ns 16 3 (2) b Q597R ER, PM 24 9 (4) b 15 8 (4) c 8 7 (2) b L693 ER 26 11 (5) a 12 6 (4) c nd C1477R PM 53 12 (9) ns 33 9 (6) b 12 2 (2) b HA819/1466 PM 54 12 (6) ns 49 7 (6) a 108 28 (4) b HA819/C1477R PM 57 15 (4) ns 68 6 (4) a 142 24 (2) b SL, subcellular localization as detected by confocal imaging and confirmed by surface biotynilation; AnnV and ApoA-I, binding of annexin V or apoA-I in cells successfully transfected with the test construct (GFP positive); St, statistical significance. Login to comment
216 This is the case of the Tangier variant W590S, whose behavior has also been documented by Fitzgerald et al. (22). Login to comment

[hide] Lack of association between increased carotid inti... Clin Chem. 2002 Nov;48(11):2066-70. Hong SH, Riley W, Rhyne J, Friel G, Miller M
Lack of association between increased carotid intima-media thickening and decreased HDL-cholesterol in a family with a novel ABCA1 variant, G2265T.
Clin Chem. 2002 Nov;48(11):2066-70., [PMID:12407001]

Abstract [show]
Low HDL-cholesterol (HDL-C) concentrations are inversely correlated with cardiovascular disease, and previous studies have demonstrated that variants in the ATP-binding cassette transporter, ABCA1, are responsible for a proportion of HDL-C deficiency states. We identified a novel variant in ABCA1 in a kindred with decreased HDL-C. This variant was not identified in >200 chromosomes of healthy individuals. The proband, a heterozygote for G2265T, developed premature coronary artery disease. In addition to low HDL-C, six biological family members heterozygous for the ABCA1 variant exhibited low HDL-C concentrations compared with unaffected family members (0.83 +/- 0.32 vs 1.33 +/- 0.36 mmol/L; P = 0.009). Despite the decreased HDL-C, carotid artery B-mode ultrasound studies failed to reveal increased intima-media thickening in affected individuals compared with age- and sex-matched controls. Although these data extend previous observations that a single defective ABCA1 allele may lead to decreased HDL-C, associated evidence of early atherosclerosis was not confirmed.

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89 Other mutations in ABCA1 associated with TD and in close proximity to W590S have been identified in amino acids 587 and 597 (3, 27). Login to comment
90 Finally, Bodzioch et al. (4) reported a mutation in an individual with TD at the same nucleotide (2265) that led to a predicted conversion of Trp to Ser (W590S). Login to comment

[hide] Naturally occurring mutations in the largest extra... J Biol Chem. 2002 Sep 6;277(36):33178-87. Epub 2002 Jun 25. Fitzgerald ML, Morris AL, Rhee JS, Andersson LP, Mendez AJ, Freeman MW
Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I.
J Biol Chem. 2002 Sep 6;277(36):33178-87. Epub 2002 Jun 25., [PMID:12084722]

Abstract [show]
The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.

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4 Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Login to comment
7 The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux. Login to comment
39 DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment
70 Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment
71 Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment
116 The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment
119 Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 Ϯ 0.04% versus 1.21 Ϯ 0.39%; WT, 3.92 Ϯ 0.13% versus 1.9 Ϯ 0.08%; R587W, 1.78 Ϯ 0.11% versus 1.61 Ϯ 0.24%; W590S, 1.92 Ϯ 0.24% versus 1.63 Ϯ 0.08%; Q597R, 1.5 Ϯ 0.14% versus 1.49 Ϯ 0.03%; C1477R, 1.67 Ϯ 0.18% versus 1.52 Ϯ 0.15%; and S1506L, 1.66 Ϯ 0.28% versus 1.6 Ϯ 0.13%. Login to comment
200 Surprisingly, the W590S mutant consistently cross-linked to apoA-I at an efficiency that was equal to or greater than that of wild type ABCA1. Login to comment
202 DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids ϳ44-640 and ϳ1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment
212 Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment
263 What is clear is that a direct interaction between apoA-I and ABCA1 is not sufficient for the movement of cholesterol out of the cell, because the W590S mutant retained full cross-linking activity and yet could not efflux cholesterol. Login to comment
37 DNA Constructs-Five missense mutants of ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) were generated using overlap polymerase chain reaction methods, as described previously (17). Login to comment
67 Missense Mutations in Two Putative Extracellular Loops of ABCA1 Ablate Efflux Activity-Five missense mutations (R587W, W590S, Q597R, C1477R, and S1506L) were introduced into a wild type ABCA1 cDNA using PCR mutagenesis techniques. Login to comment
68 Three of these mutations (R587W, W590S, and Q597R) fall in a tight cluster within the large N-terminal loop at a point near the putative second transmembrane domain shown in Fig. 3. Login to comment
112 The cells were transfected with either empty vector (mock), wild type ABCA1 (WT), or ABCA1 constructs carrying the indicated point mutations (R587W, W590S, Q597R, C1477R, and S1506L). Login to comment
115 Absolute apoA-I and medium efflux values, respectively, are as follows: mock, 1.59 afe; 0.04% versus 1.21 afe; 0.39%; WT, 3.92 afe; 0.13% versus 1.9 afe; 0.08%; R587W, 1.78 afe; 0.11% versus 1.61 afe; 0.24%; W590S, 1.92 afe; 0.24% versus 1.63 afe; 0.08%; Q597R, 1.5 afe; 0.14% versus 1.49 afe; 0.03%; C1477R, 1.67 afe; 0.18% versus 1.52 afe; 0.15%; and S1506L, 1.66 afe; 0.28% versus 1.6 afe; 0.13%. Login to comment
192 Surprisingly, the W590S mutant consistently cross-linked to apoA-I at an efficiency that was equal to or greater than that of wild type ABCA1. Login to comment
194 DISCUSSION In this study, we have established that several naturally occurring missense mutations in ABCA1 (R587W, W590S, Q597R, C1477R, and S1506L) located in the two largest loop domains of the protein (comprising amino acids b03;44-640 and b03;1371-1649, respectively) are, in fact, loss-of-function mutations. Login to comment
204 Although three of the mutations (Q597R, C1477R, and S1506L) showed no appreciable cross-linking to apoA-I, the R587W mutant had an intermediate activity, and the W590S mutant retained full, if not enhanced, cross-linking to the apoprotein. Login to comment
254 What is clear is that a direct interaction between apoA-I and ABCA1 is not sufficient for the movement of cholesterol out of the cell, because the W590S mutant retained full cross-linking activity and yet could not efflux cholesterol. Login to comment

[hide] The effects of neurological disorder-related codon... Biosci Biotechnol Biochem. 2012;76(12):2289-93. Epub 2012 Dec 7. Tomioka M, Toda Y, Kurisu J, Kimura Y, Kengaku M, Ueda K
The effects of neurological disorder-related codon variations of ABCA13 on the function of the ABC protein.
Biosci Biotechnol Biochem. 2012;76(12):2289-93. Epub 2012 Dec 7., [PMID:23221702]

Abstract [show]
Rare coding variants of ATP-binding cassette protein A13 (ABCA13) contribute to the risk of neurological disorders, but little is known about the physiological function of ABCA13 and how single nucleotide polymorphisms (SNPs) affect it. Here, we examined the effects of neurological disorder-related SNPs ABCA13, T4031A and R4843C in the context of ABCA1, and found that the former SNP (T1088A in ABCA1) severely impaired the ABCA1 functions of apolipoprotein A-I (apoA-I) binding and cholesterol efflux. The antibody against mouse ABCA13 reacted with neurons in the cerebral cortex, hippocampus, and cerebellum. These results suggest that the T4031A replacement affects the function of ABCA13 in the brain.

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56 ApoA-I-dependent free cholesterol efflux from HEK293 cells stably expressing ABCA1, non-functional mutant (MM), a Tangier mutant (W590S), and SNP mutants are shown. Login to comment
68 We also analyzed cholesterol efflux from cells expressing non-functional ABCA1-MM, in which two critical lysine residues in the NBDs were replaced with methionine residues and a Tangier disease mutant, ABCA1-W590S. Login to comment
69 When the cells were incubated with a physiological concentration of apoA-I (10 mg/mL) for 24 h, a significant amount of cholesterol was transported from them to the medium by ABCA1 (Fig. 2A), but we found that ABCA1-MM did not mediate apoA-I-dependent cholesterol efflux, and that W590S Tangier mutation reduced cholesterol efflux by about 55%, as reported previously.12) Furthermore, the T1088A SNP drastically reduced cholesterol efflux, by about 90%. Login to comment
70 However, the K2031C SNP did not significantly affect cholesterol efflux as compared to the wild type or K2031R (Fig. 2A). Login to comment
76 The total expression of T1088A was similar to that of the wild-type and the other mutants, with the exception of W590S (Fig. 3A). Login to comment
77 However, the amount of biotinylated ABCA1-T1088A was low as compared with the other mutants, and the fraction of surface localization was reduced by about 70% as compared to ABCA1-MM (Fig. 3B). Login to comment
87 A B W590S Fig. 3. Login to comment
57 ApoA-I-dependent free cholesterol efflux from HEK293 cells stably expressing ABCA1, non-functional mutant (MM), a Tangier mutant (W590S), and SNP mutants are shown. Login to comment
88 A B W590S Fig. 3. Login to comment

[hide] ABCA1 dimer-monomer interconversion during HDL gen... Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5034-9. doi: 10.1073/pnas.1220703110. Epub 2013 Mar 11. Nagata KO, Nakada C, Kasai RS, Kusumi A, Ueda K
ABCA1 dimer-monomer interconversion during HDL generation revealed by single-molecule imaging.
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5034-9. doi: 10.1073/pnas.1220703110. Epub 2013 Mar 11., [PMID:23479619]

Abstract [show]
The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATP-binding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope. We found that about 70% of total ABCA1-GFP spots are immobilized on the plasma membrane and estimated that about 89% of immobile ABCA1 molecules are in dimers. Furthermore, an ATPase-deficient ABCA1 mutant failed to be immobilized or form a dimer. We found that the lipid acceptor apoA-I interacts with the ABCA1 dimer to generate dHDL and is followed by ABCA1 dimer-monomer interconversion. This indicates that the formation of the ABCA1 dimer is the key for apoA-I binding and nascent HDL generation. Our findings suggest the physiological significance of conversion of the ABCA1 monomer to a dimer: The dimer serves as a receptor for two apoA-I molecules for dHDL particle generation.

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236 16. Nagao K, Zhao Y, Takahashi K, Kimura Y, Ueda K (2009) Sodium taurocholate-dependent lipid efflux by ABCA1: Effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation. Login to comment

[hide] Factors controlling nascent high-density lipoprote... FASEB J. 2013 Jul;27(7):2880-92. doi: 10.1096/fj.12-216564. Epub 2013 Mar 29. Lyssenko NN, Nickel M, Tang C, Phillips MC
Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability.
FASEB J. 2013 Jul;27(7):2880-92. doi: 10.1096/fj.12-216564. Epub 2013 Mar 29., [PMID:23543682]

Abstract [show]
Nascent high-density lipoprotein (HDL) particles arise in different sizes. We have sought to uncover factors that control this size heterogeneity. Gel filtration, native PAGE, and protein cross-linking were used to analyze the size heterogeneity of nascent HDL produced by BHK-ABCA1, RAW 264.7, J774, and HepG2 cells under different levels of two factors considered as a ratio, the availability of apolipoprotein AI (apoAI) -accessible cell lipid, and concentration of extracellular lipid-free apoAI. Increases in the available cell lipid:apoAI ratio due to either elevated ATP-binding cassette transporter A1 (ABCA1) expression and activity or raised cell density (i.e., increasing numerator) shifted the production of nascent HDL from smaller particles with fewer apoAI molecules per particle and fewer molecules of choline-phospholipid and cholesterol per apoAI molecule to larger particles that contained more apoAI and more lipid per molecule of apoAI. A further shift to larger particles was observed in BHK-ABCA1 cells when the available cell lipid:apoAI ratio was raised still higher by decreasing the apoAI concentration (i.e., the denominator). These changes in nascent HDL biogenesis were reminiscent of the transition that occurs in the size composition of reconstituted HDL in response to an increasing initial lipid:apoAI molar ratio. Thus, the ratio of available cell lipid:apoAI is a fundamental cause of nascent HDL size heterogeneity, and rHDL formation is a good model of nascent HDL biogenesis.

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37 MATERIALS AND METHODS Cell culture and biogenesis of nascent HDL BHK-ABCA1, BHK-ABCA1(W590S), and BHK-ABCA1(C1477R) cells expressing human wild-type or mutant ABCA1 have been previously described (36, 37). Login to comment
166 Mutations in ABCA1 that compromise its functionality shift nascent HDL production to the smaller particle species W590S and C1477R are naturally occurring mutations in human ABCA1 that impair its functionality and cause Tangier disease (49) but do not affect its subcellular localization or expression levels (37). Login to comment
167 To determine how deficiency in ABCA1 functionality affects size heterogeneity of nascent HDL particles, BHK-ABCA1, BHK-ABCA1(W590S), and BHK-ABCA1(C1477R) cells were plated at the same density, labeled with [3 H]cholesterol, induced to express ABCA1 at the maximum level, and exposed to a high concentration of human lipid-free apoAI, followed by the regular procedure to collect and analyze nascent HDL via gel filtration. Login to comment
194 BHK cells expressing wild-type ABCA1, ABCA1(W590S), or ABCA1(C1477R) were plated at the same density, labeled with [3 H]cholesterol, treated with 10 nM mifepristone, and exposed to 10 òe;g/ml of human apoAI to generate nascent HDL. Login to comment
195 The decreased size of gel-filtration elution peaks indicates that W590S and especially C1477R mutations dramatically reduced the ability of ABCA1 to facilitate nascent HDL assembly. Login to comment

[hide] ABCA1 mediates unfolding of apolipoprotein AI N te... Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1197-205. doi: 10.1161/ATVBAHA.112.301195. Epub 2013 Apr 4. Wang S, Gulshan K, Brubaker G, Hazen SL, Smith JD
ABCA1 mediates unfolding of apolipoprotein AI N terminus on the cell surface before lipidation and release of nascent high-density lipoprotein.
Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1197-205. doi: 10.1161/ATVBAHA.112.301195. Epub 2013 Apr 4., [PMID:23559627]

Abstract [show]
OBJECTIVE: To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein. APPROACH AND RESULTS: HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Apolipoprotein AI (ApoAI) binding, plasma membrane remodeling, cholesterol efflux, apoAI cell surface unfolding, and apoAI cell surface lipidation were determined, the latter 2 measured using novel fluorescent apoAI indicators. The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. However, all ABCA1 isoforms led to apoAI unfolding at the cell surface, which was higher for the isoforms that increased apoAI binding. ApoAI lipidation was not detected on ABCA1-expressing cells, only in the conditioned medium, consistent with rapid release of nascent high-density lipoprotein from ABCA1-expressing cells. CONCLUSIONS: We identified a third activity of ABCA1, the ability to unfold the N terminus of apoAI on the cell surface. Our results support a model in which unfolded apoAI on the cell surface is an intermediate in its lipidation and that, once apoAI is lipidated, it forms an unstable structure that is rapidly released from the cells to generate high-density lipoprotein.

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1 One recent model from Phillips proposes that ABCA1 shuttles phospholipids from the inner to extracellular face of the plasma membrane, resulting in membrane bulges with high curvature that are sufficient to allow apoAI penetration and nHDL formation.4 Mutations in ABCA1 lead to Tangier disease and familial hypoalphalipoproteinemia, characterized by very low levels of plasma HDL-cholesterol.5 Functional studies of certain Tangier disease mutations that are properly trafficked to the plasma membrane demonstrate that ABCA1 seems to have at least 2 distinct activities6 : apoAI binding and plasma membrane remodeling, the latter demonstrated through phosphatidylserine (PS) translocation to the outer leaflet of the plasma membrane.7 The W590S mutation in the first extracellular domain of ABCA1 is defective in PS translocation, but competent for apoAI binding.5,7,8 In contrast, the C1477R mutation in the second extracellular domain of ABCA1 is defective in apoAI binding, but competent for PS translocation.7 Further demonstration of the ability of ABCA1, and the deficit of W590S isoform, to remodel the plasma membrane was provided by Nagao et al,8 who used sodium taurocholate (NaTC) as a weak detergent extracellular lipid acceptor; wild type (WT) but not W590S ABAC1 mediates increased lipid efflux to NaTC. Login to comment
4 Approach and Results-HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Login to comment
6 The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. Login to comment
24 HEK293 cells were stably transfected with different murine ABCA1-green fluorescent protein (GFP) fusion vectors encoding WT, K939M, W590S, and C1477R isoforms, the latter 2 identified as Tangier disease-causing mutations in the first and second large extracellular domains, respectively.6 Several clonally derived cell lines from each construction were screened by confocal fluorescence microscopy and flow cytometry to select lines for further study with equivalent ABCA1 expression. Login to comment
25 As previously described,5,9 WT, W590S, C1477R, and K939M ABCA1-GFP fusions were processed correctly in cells and expressed on the plasma membrane (Figure 1A). Login to comment
28 We confirmed the previously identified apoAI-binding activity of the WT and W590S-ABCA1 isoforms, with both having ࣈ6-fold higher apoAI binding than the control cells (P<0.001 by ANOVA posttest). Login to comment
33 The W590S cells had a small 1.26-fold increase in cell surface PS (P<0.05 versus control by ANOVA posttest), whereas the K939M-ABCA1 isoform had no cell surface PS increase (Figure 2B). Login to comment
34 Thus, our results confirmed previous findings by Singaraja et al5 and Nagao et al8 that the W590S mutation in ABCA1`s first extracellular domain greatly diminishes PS floppase activity indicative of a defect in plasma membrane remodeling, whereas the C1477R mutation in its second large extracellular domain abolishes apoAI binding. Login to comment
36 In the absence of any acceptor, WT ABCA1 increased basal 3 H cholesterol efflux by 32% (P<0.05 by ANOVA posttest versus HEK), which has previously been shown to be attributable to increased microparticle release.14 The C1477R-ABCA1 isoform also had increased basal cholesterol efflux activity (34% increase; P<0.05); however, efflux from the W590S-ABCA1 isoform cell line was not significantly different from the control HEK cells. Login to comment
39 A, Confocal microscopy of wild type (WT), W590S, C1477R, and K939M ABCA1-GFP isoforms shows cell surface expression. Login to comment
40 B, Similar expression levels of WT, W590S, C1477R, and K939M ABCA1-GFP cell lines shown by flow cytometry (n=3; mean&#b1;SD; different numbers above the bars show P<0.001, by ANOVA posttest). Login to comment
42 Interestingly, both Tangier disease mutations supported partial efflux to apoAI with 4.27and 4.02-fold increases above the control HEK cells for the W590S and C1477R isoforms, respectively. Login to comment
43 All 4 cell lines have significantly different efflux to apoAI (P<0.001 by ANOVA posttest), except for efflux from the W590S and C1477R cells that were not different from each other. Login to comment
46 The W590S-ABCA1 isoform, which can mediate binding of apoAI, yielded a similar cholesterol efflux as the HEK cells at 2.57%. Login to comment
49 Thus, our findings show that ABCA1 mutations that disrupt either plasma membrane remodeling (W590S) or apoAI binding (C1477R) are still competent to mediate cholesterol efflux to apoAI, albeit at reduced efficiency. Login to comment
62 A, Alexa647-labeled apoAI binding to nontransfected cells (HEK) and cells stably transfected with wild type (WT), W590S, C1477R, and K939M ABCA1-green fluorescent protein (GFP) vectors assayed by flow cytometry (n=3; mean&#b1;SD; P<0.0005 for HEK in the presence or absence of labeled apoAI by t test; for the 5 cell types in the presence of labeled apoAI different numbers above the bars show P<0.001; by ANOVA posttest). Login to comment
64 C, Cholesterol efflux from control HEK cells and WT, W590S, and C1477R ABCA1-GFP transfected cells in the absence of exogenous acceptors or in the presence of 5 bc;g/mL apoAI or 2 mmol/L sodium taurocholate (NaTC; n=3; mean&#b1;SD; different numbers above the bars show P<0.05, by ANOVA posttest). Login to comment
71 However, on incubation of this indicator with the WT and W590S ABCA1-expressing cells, there was a rightward shift to a higher cellular Bodipy-TMR/Alexa647 ratio peak of 1.3 to 1.4 (1.86-2.0-fold increase). Login to comment
74 The partially active W590S isoform has 2 activities: apoAI binding and apoAI unfolding. Login to comment
92 The W590S and C1447R mutant isoforms had intermediate ratios ࣈ1.4 and 1.2-fold higher than that observed from the control HEK cells (P<0.001 and P<0.05, respectively, versus control cells by ANOVA posttest). Login to comment
94 We attribute the intermediate fluorescence ratio levels in the conditioned media from the W590S and C1447R transfected cells to be a result of fewer lipidated particles released into the conditioned media, rather than to less unfolding of apoAI per particle. Login to comment
113 We chose to further study 2 specific mutations in the first (W590S) and second (C1477R) extracellular domains, respectively, based on their previously identified distinct activities. Login to comment
114 Fitzgerald et al17 examined 5 Tangier disease mutations that mapped to the 2 large extracellular domains, and reported that only the W590S mutation in the first extracellular domain was still competent to mediate apoAI cross-linking, whereas other mutations in the first (R587W and Q597R) and second (C1477R and S1506L) extracellular domains could not mediate apoAI cross-linking. Login to comment
115 Although the flag-tagged R587W and Q597R variants were reported to be expressed on the plasma membrane in transfected cells,17 2 other independent groups reported that these 2 variants have impaired processing and decreased cell surface expression5,8,18 ; but all agree that the W590S is expressed on the plasma membrane similarly to the WT isoform and can mediate apoAI binding. Login to comment
116 Our findings reproduce that the W590S isoform has plasma membrane localization and WT levels of apoAI-binding activity; and that it has partial cholesterol efflux activity to extracellular apoAI compared with the WT isoform, as previously demonstrated.5,7,8,18 We chose to study the C1477R mutation in the second extracellular domain because it is processed correctly to the plasma membrane and has defective apoAI binding,5,7,17 but it retains its PS translocase activity and partial efflux activity,7 all findings that we confirmed in the current study, where we found ࣈ50% cholesterol efflux activity to apoAI. Login to comment
117 Nagao et al8 first demonstrated the use of NaTC as a nonpeptide acceptor of cellular lipids, and that WT ABCA1 increased lipid efflux to this weak detergent, and that the W590S mutation abolished this activity. Login to comment
118 Here, we compared the acceptor activity of NaTC for cells expressing WT, W590S, and C1477R-ABCA1 isoforms, and found that the C1477R mutant has equivalent efflux to NaTC as the WT isoform, whereas the W590S mutant has no detectable efflux to NaTC above nontransfected cells. Login to comment
119 The NaTC efflux activities of these ABCA1 isoforms is similar to the PS translocase activity, and thus both of these assays are evidence that the WT and C1477R isoforms can remodel the plasma membrane, whereas the W590S isoform is mostly deficient in this activity. Login to comment
130 Through the use of an N-terminal apoAI unfolding indicator, we observed what we estimate to be ࣈ75% unfolding of apoAI on the surface of HEK cells expressing either WT or W590S-ABCA1 isoforms, both of which have full apoAI-binding activity (Figure 4A). Login to comment
131 The slight rightward shift of the W590S isoform may be as a result of defective membrane remodeling in the W590S-expressing cells, leading to the unfolded apoAI having fewer opportunities to form rHDL and be released from the cell. Login to comment
138 The presence of the high-affinity apoAI-binding site in WT and W590S-ABCA1 isoforms would promote apoAI proximity to the low-affinity binding site and therefore increase apoAI unfolding. Login to comment
149 A model for the mechanism of ABCA1 action from Phillips states that phospholipid translocation produces membrane protuberances that in themselves because of their small radius and surface packing are sufficient to spontaneously interact with apoAI and form nHDL.4 However, the observation that the W590S-ABCA1 isoform is not competent for phospholipid translocation and membrane remodeling but is still able to mediate HDL assembly, albeit at reduced efficiency, does not support this model. Login to comment
161 We propose the steps through formation of the LC state may be reversible, and there is experimental evidence that W590S ABCA1-expressing cells bind and release nonlipidated apoAI from the cell,30 and we show here that the apoAI bound by this mutant isoform is also in the U state. Login to comment

[hide] Differential phospholipid substrates and direction... J Biol Chem. 2013 Nov 29;288(48):34414-26. doi: 10.1074/jbc.M113.508812. Epub 2013 Oct 4. Quazi F, Molday RS
Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants.
J Biol Chem. 2013 Nov 29;288(48):34414-26. doi: 10.1074/jbc.M113.508812. Epub 2013 Oct 4., [PMID:24097981]

Abstract [show]
ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.

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65 Mutations introduced by overlap extension PCR using Pfu AD DNA polymerase in ABCA1 included S100C, W590S, F593L, N935S, T929I, C1477R, T1512M, R2081W, and P2150L. Login to comment
67 ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment
208 Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment
229 Variants in the ECD1 (S100C, W590S, and F593L), NBD1 (T929I and N935S), and NBD2 (R2081W) of ABCA1 showed significantly reduced ATPase activities in the range of 20-35% of WT activity (Fig. 7A). Login to comment
232 ABCA4 variants showed a similar ATPase activity profile as the ABCA1 mutants with the exception of the T1537M mutation of ABCA4, which was significantly lower than the corresponding T1512M mutant in ABCA1. Login to comment
251 Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment
295 The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (20-30% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment
211 Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment
254 Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment
298 The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (20-30% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment
64 Mutations introduced by overlap extension PCR using Pfu AD DNA polymerase in ABCA1 included S100C, W590S, F593L, N935S, T929I, C1477R, T1512M, R2081W, and P2150L. Login to comment
206 Expression and Purification of Disease-causing ABCA1 and ABCA4 Mutants-As part of this study, we have generated a number of disease-causing mutations in ABCA1 and ABCA4 to determine their effect on the expression and functional properties of these transporters. We focused our studies on nine missense mutations in ABCA1 known to cause Tangier disease, including three (S100C, W590S, and F593L) in ECD1, two (T929I and N935S) in the NBD1, two (C1477R and T1512M) in ECD2, one (R2081W) in NBD2, and one (P2150L) in the C-terminal segment as shown in Fig. 6A (blue). Login to comment
227 Variants in the ECD1 (S100C, W590S, and F593L), NBD1 (T929I and N935S), and NBD2 (R2081W) of ABCA1 showed significantly reduced ATPase activities in the range of 20-35% of WT activity (Fig. 7A). Login to comment
249 Some disease alleles such as W590S, C1477R, and P2150L (ABCA1) have conserved residues in ABCA4, but mutations in these positions in ABCA4 have yet to be linked to Stargardt disease. Login to comment
293 The ABCA1/ABCA4 mutants in the ECD1 (S100C/S100P, W590S/ W605S, and F593L/F608L) displayed the lowest activities (2030% WT), whereas those in the ECD2 (C1477R/C1502R and T1512M/T1537M) and the P2150L/P2180L mutants in the C terminus showed the highest activities (60-100% WT). Login to comment

[hide] Sphingomyelin depletion impairs anionic phospholip... J Biol Chem. 2013 Dec 27;288(52):37166-79. doi: 10.1074/jbc.M113.512244. Epub 2013 Nov 12. Gulshan K, Brubaker G, Wang S, Hazen SL, Smith JD
Sphingomyelin depletion impairs anionic phospholipid inward translocation and induces cholesterol efflux.
J Biol Chem. 2013 Dec 27;288(52):37166-79. doi: 10.1074/jbc.M113.512244. Epub 2013 Nov 12., [PMID:24220029]

Abstract [show]
The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.

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5 The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Login to comment
8 Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Login to comment
22 The W590S Tangier mutant allele of ABCA1 retains its ability to bind lipid-free lipoprotein apoAI, but has impaired PS floppase activity and efflux of phospholipids and cholesterol to apoAI (15, 24-26). Login to comment
38 We found that SM depletion by myriocin or sphingomyelinase (SMase) treatment could compensate for the defective PS floppase activity of the mutant W590S-ABCA1 isoform, restoring its cholesterol efflux activity to apoAI. Login to comment
105 RESULTS Myriocin Enhances Cholesterol Efflux and Restores Efflux Activity of the W590S Abca1 Mutation-We assessed the effects of myriocin, a potent inhibitor of the first step in sphingolipid synthesis on cholesterol efflux to apoAI, a process that is mediated by ABCA1, in stably transfected HEK293 cells expressing equivalent levels of wild type (WT) murine and two mutant ABCA1 isoforms (15). Login to comment
107 As previously observed (15, 24-26), cholesterol efflux to apoAI from cells expressing either the W590S (PS translocation deficient) or C1477R (apoAI binding deficient) ABCA1 mutant isoforms was partially, but not completely impaired compared with the WT isoform (p b0d; 0.001 compared with control HEK and WT ABCA1 by ANOVA post test). Login to comment
108 Upon treatment with 10 òe;M myriocin, cholesterol efflux was significantly increased in all four cell types, however, the net increase was greatest for the W590S mutant isoform (2.34% increase, 1.72-fold), with the wild type ABCA1 isoform having a 1.44% net increase, and the other two cell lines having b0d;1% net increases. Login to comment
109 Thus, myriocin treatment could overcome much of the impaired cholesterol efflux activity of the W590S Abca1 mutation, although not greatly improving efflux from the C1477R mutation. Login to comment
123 We then compared the effects of depleting sphingomyelin on the cholesterol efflux activity of the WT and W590S isoforms of ABCA1 in stably transfected HEK cells. Login to comment
124 Increasing levels of SMase led to increased cholesterol efflux to apoAI for both isoforms, such that at the highest dose of SMase, the W590S isoform had as much efflux activity as the WT isoform in the absence of SMase (Fig. 3G). Login to comment
125 Thus, we could obtain complete restoration of the efflux activity of the ABCA1 W590S isoform FIGURE 1. Login to comment
127 A, myriocin compensates for the W590S-Abca1 mutation. Login to comment
128 Non-transfected HEK293 cells along with HEK cells transfected with WT-Abca1-GFP, W590S-Abca1-GFP, or C1477R-Abca1-GFP isoforms were incubated with radiolabeled cholesterol and subsequently chased with apoAI (5 òe;g/ml) for 6 h. Login to comment
142 Taken together, our data strongly indicate that Sphingomyelin Depletion Impairs Phospholipid Flip 37170 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 52ߦDECEMBER 27, 2013 decreased SM, rather than increased ceramide, is responsible for the induction of cholesterol efflux, which can compensate for the impaired PS translocation in the W590S ABCA1 mutation. Login to comment
147 Although WT ABCA1 expression increased cholesterol efflux to NaTC to almost 6-fold, expression of the W590S ABCA1 isoform increased efflux to NaTC by only 3.1-fold, as previously observed (15, 25). Login to comment
148 Myriocin increased cholesterol efflux from cells expressing either ABCA1 isoforms (1.6-fold for the WT and 2.1-fold for the W590S isoform), such that the myriocin-treated W590S-expressing cells exhibited comparable efflux to NaTC as seen in the myriocin-untreated WT-ABCA1 expressing cells (see gray bars in Fig. 4B). Login to comment
149 Thus, the SM depletion effect on cholesterol efflux is observed both in the absence and presence of ABCA1 expression, and can compensate for the W590S Abca1 mutation even with a non-selective acceptor. Login to comment
151 Because the ABCA1 W590S mutation has defective translocation of PS to the outer leaflet of the plasma membrane (11, 24), we assessed whether SM depletion altered the translocation of phospholipids across the plasma membrane. Login to comment
154 As reported earlier, the W590S-Abca1 expressing HEK cells showed decreased cell surface PS as compared with the WT isoform (15, 24, 25), only 1.3-fold higher than control HEK cells. Login to comment
173 HEK cells stably transfected with either WT-Abca1-GFP or W590S-Abca1-GFP isoforms were loaded with radiolabeled cholesterol and chased with apoAI (5 òe;g/ml) for 6 h. Login to comment
180 HEK293 cells or stably transfected lines with either WT or W590S isoform of Abca1 were incubated with radiolabeled cholesterol and subsequently treated with or without 10 òe;M myriocin for 12 h. Login to comment
184 Here, different letters above the bars show p b0d; 0.01 by ANOVA with a Bonferroni post test. Sphingomyelin Depletion Impairs Phospholipid Flip DECEMBER 27, 2013ߦVOLUME 288ߦNUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 37171 at SEMMELWEIS UNIV OF MEDICINE on December , ment of the W590S-ABAC1 cells with myriocin, there was an almost complete restoration of cell surface PS (1.55-fold versus control HEK cells) (Fig. 5, A and B). Login to comment
188 B, primary flow cytometry data showing shift in annexin V signal in HEK293, HEK-Abca1, and HEK-W590S-Abca1 cells with or without treatment with myriocin. Login to comment
290 This was also indicated by our finding that myriocin increased cholesterol efflux to NaTC in HEK cells, but even more so in cells transfected with WT and particularly the W590S mutant isoform of ABCA1 (Fig. 4B). Login to comment
292 In fact, ABCA1 itself also raises cell surface PS, although this activity is greatly diminished in the W590S ABCA1 mutation that leads to Tangier disease (15, 24-26). Login to comment
293 The SM depletion-mediated increase in cell surface PS was sufficient to compensate for the defective PS translocation in the W590S-Abca1 expressing HEK cells, restoring cholesterol efflux to apoAI (Fig. 3G) and NaTC (Fig. 4B), providing direct proof that the ABCA1 PS floppase activity plays a role in ABCA1-mediated cholesterol efflux. Login to comment
297 This increase in cell surface PS could partially restore the activity of the PS-floppase-deficient Abca1 W590S mutation (Figs. Login to comment

[hide] HDL biogenesis, remodeling, and catabolism. Handb Exp Pharmacol. 2015;224:53-111. doi: 10.1007/978-3-319-09665-0_2. Zannis VI, Fotakis P, Koukos G, Kardassis D, Ehnholm C, Jauhiainen M, Chroni A
HDL biogenesis, remodeling, and catabolism.
Handb Exp Pharmacol. 2015;224:53-111. doi: 10.1007/978-3-319-09665-0_2., [PMID:25522986]

Abstract [show]
In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.

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50 A notable exemption is the ABCA1[W590S] mutant which cross-linked stronger to apoA-I than to WT ABCA1 but had diminished capacity to promote cholesterol efflux and to promote formation of HDL (Bodzioch et al. 1999; Fitzgerald et al. 2001, 2002). Login to comment

[hide] The effect of phospholipid composition of reconsti... J Lipid Res. 2015 Sep;56(9):1727-37. doi: 10.1194/jlr.M060285. Epub 2015 Jun 27. Schwendeman A, Sviridov DO, Yuan W, Guo Y, Morin EE, Yuan Y, Stonik J, Freeman L, Ossoli A, Thacker S, Killion S, Pryor M, Chen YE, Turner S, Remaley AT
The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties.
J Lipid Res. 2015 Sep;56(9):1727-37. doi: 10.1194/jlr.M060285. Epub 2015 Jun 27., [PMID:26117661]

Abstract [show]
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 prebeta HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of prebeta formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-alpha, IL-6, and IL-1beta 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.

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277 The comparison of in vitro cholesterol efflux by ApoA-I and ApoA-I-Milano formulated with either 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) or SM was conducted in Chinese hamster ovary cells, J774 macrophages, and BHK cells transfected with ABCA1, nonfunctional ABCA1 mutant (W590S), and ABCG1 (9). Login to comment

[hide] Update on the molecular biology of dyslipidemias. Clin Chim Acta. 2015 Nov 4. pii: S0009-8981(15)30036-X. doi: 10.1016/j.cca.2015.10.033. Ramasamy I
Update on the molecular biology of dyslipidemias.
Clin Chim Acta. 2015 Nov 4. pii: S0009-8981(15)30036-X. doi: 10.1016/j.cca.2015.10.033., [PMID:26546829]

Abstract [show]
Dyslipidemia is a commonly encountered clinical condition and is an important determinant of cardiovascular disease. Although secondary factors play a role in clinical expression, dyslipidemias have a strong genetic component. Familial hypercholesterolemia is usually due to loss-of-function mutations in LDLR, the gene coding for low density lipoprotein receptor and genes encoding for proteins that interact with the receptor: APOB, PCSK9 and LDLRAP1. Monogenic hypertriglyceridemia is the result of mutations in genes that regulate the metabolism of triglyceride rich lipoproteins (eg LPL, APOC2, APOA5, LMF1, GPIHBP1). Conversely familial hypobetalipoproteinemia is caused by inactivation of the PCSK9 gene which increases the number of LDL receptors and decreases plasma cholesterol. Mutations in the genes APOB, and ANGPTL3 and ANGPTL4 (that encode angiopoietin-like proteins which inhibit lipoprotein lipase activity) can further cause low levels of apoB containing lipoproteins. Abetalipoproteinemia and chylomicron retention disease are due to mutations in the microsomal transfer protein and Sar1b-GTPase genes, which affect the secretion of apoB containing lipoproteins. Dysbetalipoproteinemia stems from dysfunctional apoE and is characterized by the accumulation of remnants of chylomicrons and very low density lipoproteins. ApoE deficiency can cause a similar phenotype or rarely mutations in apoE can be associated with lipoprotein glomerulopathy. Low HDL can result from mutations in a number of genes regulating HDL production or catabolism; apoAI, lecithin: cholesterol acyltransferase and the ATP-binding cassette transporter ABCA1. Patients with cholesteryl ester transfer protein deficiency have markedly increased HDL cholesterol. Both common and rare genetic variants contribute to susceptibility to dyslipidemias. In contrast to rare familial syndromes, in most patients, dyslipidemias have a complex genetic etiology consisting of multiple genetic variants as established by genome wide association studies. Secondary factors, obesity, metabolic syndrome, diabetes, renal disease, estrogen and antipsychotics can increase the likelihood of clinical presentation of an individual with predisposed genetic susceptibility to hyperlipoproteinemia. The genetic profiles studied are far from complete and there is room for further characterization of genes influencing lipid levels. Genetic assessment can help identify patients at risk for developing dyslipidemias and for treatment decisions based on 'risk allele' profiles. This review will present the current information on the genetics and pathophysiology of disorders that cause dyslipidemias.

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1058 The mutant W590S has a near-normal apolipoprotein binding activity but defective lipid transport (471). Login to comment