ABCMdb

ABCA1 p.Gln597Arg

ClinVar:	c.1790A>G , p.Gln597Arg D , Pathogenic
Predicted by SNAP2:	A: D (80%), C: D (80%), D: D (85%), E: D (80%), F: D (91%), G: D (80%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (75%), N: D (80%), P: D (91%), R: D (91%), S: D (80%), T: D (75%), V: D (80%), W: D (91%), Y: D (85%),
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, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[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] Anticancer Activity of the Cholesterol Exporter AB... Cell Rep. 2012 Sep 27;2(3):580-90. doi: 10.1016/j.celrep.2012.08.011. Epub 2012 Sep 13. Smith B, Land H
Anticancer Activity of the Cholesterol Exporter ABCA1 Gene.
Cell Rep. 2012 Sep 27;2(3):580-90. doi: 10.1016/j.celrep.2012.08.011. Epub 2012 Sep 13., [PMID:22981231]

Abstract [show]
The ABCA1 protein mediates the transfer of cellular cholesterol across the plasma membrane to apolipoprotein A-I. Loss-of-function mutations in the ABCA1 gene induce Tangier disease and familial hypoalphalipoproteinemia, both cardiovascular conditions characterized by abnormally low levels of serum cholesterol, increased cholesterol in macrophages, and subsequent formation of vascular plaque. Increased intracellular cholesterol levels are also frequently found in cancer cells. Here, we demonstrate anticancer activity of ABCA1 efflux function, which is compromised following inhibition of ABCA1 gene expression by oncogenic mutations or cancer-specific ABCA1 loss-of-function mutations. In concert with elevated cholesterol synthesis found in cancer cells, ABCA1 deficiency allows for increased mitochondrial cholesterol, inhibits release of mitochondrial cell death-promoting molecules, and thus facilitates cancer cell survival, suggesting that elevated mitochondrial cholesterol is essential to the cancer phenotype.

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124 To this end, we utilized two missense mutations of human ABCA1 proteins (Q597R and C1477R) linked to TD and familial hypoalphalipoproteinemia (FHA), which were previously characterized to have diminished cholesterol efflux activity (Singaraja et al., 2006). Login to comment

[hide] Increased risk of coronary artery disease in Cauca... Biochim Biophys Acta. 2012 Mar;1821(3):416-24. doi: 10.1016/j.bbalip.2011.08.006. Epub 2011 Aug 19. Tietjen I, Hovingh GK, Singaraja R, Radomski C, McEwen J, Chan E, Mattice M, Legendre A, Kastelein JJ, Hayden MR
Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT.
Biochim Biophys Acta. 2012 Mar;1821(3):416-24. doi: 10.1016/j.bbalip.2011.08.006. Epub 2011 Aug 19., [PMID:21875686]

Abstract [show]
Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc <10th percentile but no other major lipid abnormalities, including 89 with >/=1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p=2.44 *10(-5)). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc <10th percentile. Furthermore, only mutation carriers with HDLc <5th percentile had elevated risk of CAD (odds ratio (OR)=2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p=0.05; OR=2.50 for 26 APOA1 mutation carriers, p=0.04; OR=3.44 for 38 LCAT mutation carriers, p=1.1 *10(-3)). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain >40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. 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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117 COOHA B T929I H2N R587W B A M1091T C1477R K776N N935S S1181F IVS24+1 G>C V2244I R282X D571G M640L S930F M968T R1615WIVS16-5 CA>del ABCA1 Transmembrane domain ATP-binding domain Q597R A) AA 1 AA 267 K130del L202P 74 90 98 112 122 145 167 189 211 215 233 253 APOA1 Negative charge domain Alpha-helix E222K E134DT37M 140 178 206 41127 104 121 165 200 229 360 391 Y135N V246F 127 206 369 401 Catalytic triad R322C L338H V371MV52M Y107X A117T T147I V333M Phe Leu Asp His AA 1 AA 440 R159Q I202T LCAT Alpha helixBeta sheet B) 419I.Tietjenetal./BiochimicaetBiophysicaActa1821(2012)416-424 3.4. Login to comment
169 Effects of some ABCA1 mutations described here are also consistent with previous in vitro cellular efflux studies; for example, the highly expressive ABCA1 mutations R587W, Q597R, N935S, and M1091T cause ~10-25% of wild-type efflux in vitro, while the less expressive mutations K776N and T929I cause 62 and 80% efflux, respectively [35-37]. Login to comment

[hide] Nascent HDL formation in hepatocytes and role of A... J Lipid Res. 2012 Mar;53(3):446-55. Epub 2011 Dec 20. Ji A, Wroblewski JM, Cai L, de Beer MC, Webb NR, van der Westhuyzen DR
Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI.
J Lipid Res. 2012 Mar;53(3):446-55. Epub 2011 Dec 20., [PMID:22190590]

Abstract [show]
To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.

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61 The generation of such nascent HDL particles is dependent on ABCA1 because cells lacking ABCA1 or expressing an inactive ABCA1 mutant (Q597R) were unable to form such particles (14-17). Login to comment

[hide] Membrane microdomains modulate oligomeric ABCA1 fu... J Lipid Res. 2011 Nov;52(11):2043-55. Epub 2011 Aug 16. Iatan I, Bailey D, Ruel I, Hafiane A, Campbell S, Krimbou L, Genest J
Membrane microdomains modulate oligomeric ABCA1 function: impact on apoAI-mediated lipid removal and phosphatidylcholine biosynthesis.
J Lipid Res. 2011 Nov;52(11):2043-55. Epub 2011 Aug 16., [PMID:21846716]

Abstract [show]
Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the "high-capacity binding site" (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation. Here, using sucrose gradient fractionation, we obtained evidence that both ABCA1 and [(1)(2)(5)I]apoA-I associated with the HCBS were found localized to nonraft microdomains. Interestingly, phosphatidylcholine (PtdCho) was selectively removed from nonraft domains by apoA-I, whereas sphingomyelin and cholesterol were desorbed from both detergent-resistant membranes and nonraft domains. The modulatory role of cholesterol on apoA-I binding to ABCA1/HCBS was also examined. Loading cells with cholesterol resulted in a drastic reduction in apoA-I binding. Conversely, depletion of membrane cholesterol by methyl-beta-cyclodextrin treatment resulted in a significant increase in apoA-I binding. Finally, we obtained evidence that apoA-I interaction with ABCA1 promoted the activation and gene expression of key enzymes in the PtdCho biosynthesis pathway. Taken together, these results provide strong evidence that the partitioning of ABCA1/HCBS to nonraft domains plays a pivotal role in the selective desorption of PtdCho molecules by apoA-I, allowing an optimal environment for cholesterol release and regeneration of the PtdCho-containing HCBS. This process may have important implications in preventing and treating atherosclerotic cardiovascular disease.

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64 Fibroblasts obtained from patients with Tangier disease (TD; homozygous for Q597R at the ABCA1 gene) were used as a negative control throughout the study as previously described (9, 17). Login to comment
95 ABCA1 mutant (Q597R) associated with TD was used as a negative control for binding specificity. Login to comment
125 Normal and ABCA1 mutant (Q597R) human fibroblasts (B-D) and THP-1 cells (E-G) stimulated or not with 22OH/9CRA were incubated with 10 ␮g/ml [ 125 I]apoA-I for 45 min at 37°C. After washing to remove unbound [ 125 I] apoA-I, cross-linking with DSP was performed as described in Experimental Procedures. Login to comment
151 An ABCA1 mutant (Q597R) used as a negative control for binding specificity did not show significant [125 I]apoA-I association with any sucrose fraction (Fig. 1B). Login to comment
212 An ABCA1 mutant (Q597R) that has been shown to have no significant apoA-I binding was used as a negative control. Login to comment
227 Incubation with apoA-I did not upregulate mRNA expression of CCT␣ or CK␣ in ABCA1 mutant (Q597R) fibroblasts (Fig. 5D). Login to comment
252 (A, B) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated or not with 20 ␮g/ml apoA-I for 16 h. Cells were then pulsed with 10 ␮Ci/ml methyl[ 3 H]choline for 1 h, and the distribution of radioactivity among metabolites (Cho, Pcho, and CDP-cho) and PtdCho was determined as described in Experimental Procedures. Values shown are means ± SD of triplicate measures. Login to comment
254 (C, D) Stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated or not with 20 ␮g/ml apoA-I for 16 h, followed by mRNA extraction using the RNeasy mini RNA extraction kit (Qiagen). Login to comment
270 (Top) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated in the presence of 10 ␮g/ml of [ 125 I]apoA-I for 45 min at 37°C. After washing to remove unbound [ 125 I]apoAI, DMEM was added, and plates were immediately incubated at 37°C or 4°C. Login to comment
65 Fibroblasts obtained from patients with Tangier disease (TD; homozygous for Q597R at the ABCA1 gene) were used as a negative control throughout the study as previously described (9, 17). Login to comment
96 ABCA1 mutant (Q597R) associated with TD was used as a negative control for binding specificity. Login to comment
128 Normal and ABCA1 mutant (Q597R) human fibroblasts (B-D) and THP-1 cells (E-G) stimulated or not with 22OH/9CRA were incubated with 10 g/ml [ 125 I]apoA-I for 45 min at 37&#b0;C. After washing to remove unbound [ 125 I] apoA-I, cross-linking with DSP was performed as described in Experimental Procedures. Login to comment
155 An ABCA1 mutant (Q597R) used as a negative control for binding specificity did not show significant [125 I]apoA-I association with any sucrose fraction (Fig. 1B). Login to comment
216 An ABCA1 mutant (Q597R) that has been shown to have no significant apoA-I binding was used as a negative control. Login to comment
232 Incubation with apoA-I did not upregulate mRNA expression of CCT or CK in ABCA1 mutant (Q597R) fibroblasts (Fig. 5D). Login to comment
257 (A, B) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated or not with 20 g/ml apoA-I for 16 h. Cells were then pulsed with 10 Ci/ml methyl[ 3 H]choline for 1 h, and the distribution of radioactivity among metabolites (Cho, Pcho, and CDP-cho) and PtdCho was determined as described in Experimental Procedures. Values shown are means &#b1; SD of triplicate measures. Login to comment
259 (C, D) Stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated or not with 20 g/ml apoA-I for 16 h, followed by mRNA extraction using the RNeasy mini RNA extraction kit (Qiagen). Login to comment
275 (Top) Normal and ABCA1 mutant (Q597R) fibroblasts were stimulated with 22OH/9CRA and incubated in the presence of 10 g/ml of [ 125 I]apoA-I for 45 min at 37&#b0;C. After washing to remove unbound [ 125 I]apoAI, DMEM was added, and plates were immediately incubated at 37&#b0;C or 4&#b0;C. Login to comment

[hide] Adenosine-triphosphate-binding cassette transporte... Trends Cardiovasc Med. 2010 Feb;20(2):41-9. Kang MH, Singaraja R, Hayden MR
Adenosine-triphosphate-binding cassette transporter-1 trafficking and function.
Trends Cardiovasc Med. 2010 Feb;20(2):41-9., [PMID:20656214]

Abstract [show]
Mutations in the adenosine-triphosphate-binding cassette transporter-1 (ABCA1) lead to Tangier disease, a genetic disorder characterized by an almost complete absence of plasma high-density lipoprotein cholesterol. Although the importance of ABCA1 localization to its cholesterol efflux function has been extensively characterized, the cellular itinerary of ABCA1 leading to the plasma membrane is not fully elucidated. This review will summarize the current knowledge of ABCA1 trafficking and its relationship to function. Understanding these crucial processes provides potential novel therapeutic targets to regulate high-density lipoprotein biogenesis through influencing pathways of ABCA1 trafficking.

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40 The importance of ABCA1 localization to the PM is observed in several mutations underlying TD (R587W, Q597R, ΔL693, M1091T), where mislocalization of ABCA1 away from the cell surface leads to a significant reduction in cholesterol efflux function (Singaraja et al. 2006). Login to comment
127 A number of clinically relevant mutations of ABCA1 (R587W, Q597R, ΔL693, N935H) acquire only the core and not the complex oligosaccharide chain and fail to exit the ER (Singaraja et al. 2006). Login to comment
128 A number of clinically relevant mutations of ABCA1 (R587W, Q597R, ƊL693, N935H) acquire only the core and not the complex oligosaccharide chain and fail to exit the ER (Singaraja et al. 2006). 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
54 The rank order of severity in impaired apoA-I-mediated cholesterol efflux was Q597R.A937V.R587W.C1477R.V399A.I1517R. Login to comment
93 The two most severe mutations, which reduced apoA-I-mediated lipid efflux to less than 20% of normal, were located in the first extracellular loop (Q597R) and the ATP binding site (A937V). Login to comment
96 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
97 In contrast, Singaraja et al. (14) and Tanaka et al. (25) reported that ABCA1 with the R587W or Q597R mutations had impaired translocation to the plasma membrane in transfected cells. Login to comment
99 Tanaka et al. (26) showed that endoplasmic reticulum stress can promote translocation of the Q597R ABCA1 mutant to the plasma membrane, where it still has impaired function, indicating that decreased trafficking alone cannot explain the dysfunctional effects of this mutation. Login to comment
92 The two most severe mutations, which reduced apoA-I-mediated lipid efflux to less than 20% of normal, were located in the first extracellular loop (Q597R) and the ATP binding site (A937V). Login to comment
95 Fitzgerald et al. (24) reported similar cell surface localizations for mutants R587W, W590S, Q597R, and C1477R. Login to comment
98 Tanaka et al. (26) showed that endoplasmic reticulum stress can promote translocation of the Q597R ABCA1 mutant to the plasma membrane, where it still has impaired function, indicating that decreased trafficking alone cannot explain the dysfunctional effects of this mutation. Login to comment

[hide] Effect of ABCA1 mutations on risk for myocardial i... Curr Atheroscler Rep. 2008 Oct;10(5):413-26. Iatan I, Alrasadi K, Ruel I, Alwaili K, Genest J
Effect of ABCA1 mutations on risk for myocardial infarction.
Curr Atheroscler Rep. 2008 Oct;10(5):413-26., [PMID:18706283]

Abstract [show]
The adenosine triphosphate-binding cassette A1 (ABCA1) gene codes for a cellular phospholipid and cholesterol transporter that mediates the initial and essential step in high-density lipoprotein (HDL) biogenesis: the formation of nascent HDL particles. Mutations at the ABCA1 gene locus cause severe familial HDL deficiency and, in the homozygous form, cause Tangier disease. Several studies have investigated the influence of ABCA1 variation on lipid metabolism and coronary heart disease, but they have resulted in controversial and inconsistent results. Genetic variability at the ABCA1 gene has also been associated with increased risk of myocardial infarction. In one study, this association was independent of HDL cholesterol levels, raising the possibility that the measurement of HDL cholesterol levels may not provide adequate information on the functional roles of HDL particles. Nevertheless, genomic screening for complex diseases, such as coronary heart disease, and HDL deficiency in particular, may not add additional information to that gained from conventional global cardiovascular risk stratification.

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119 Genetic variation in ABCA1 and risk of myocardial infarction Study* / year Population screened HDL-C, mmol/L ABCA1 variants Conclusions Clee et al. [28] / 2000 Within 11 TD families: Del L693, R2144X † , Del E,D1893,94 † , R909X, M1091T † , P2150L † , ivs25+1G→C, Del C6825→2145X, CTC6952- 4TT→2203X, C1477R, Q597R, T929I ABCA1 heterozygous patients had a 40%-45% decrease in HDL-C and a greater than threefold increased risk of CHD versus unaffected individuals. Login to comment

[hide] Formation of cholesterol-enriched structures by ab... Genes Cells. 2008 Aug;13(8):889-904. Tanaka AR, Kano F, Yamamoto A, Ueda K, Murata M
Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1.
Genes Cells. 2008 Aug;13(8):889-904., [PMID:18782226]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1) is a key transporter associated with excess cellular lipid efflux. Here, we report that in HEK293 cells ABCA1 functions in intracellular compartments along the endocytic pathway. Inhibition of ABCA1-GFP degradation with proteasome inhibitors induced the internalization of ABCA1 and the formation of intracellular round-shaped structures, designated "A1 bodies". Importantly, cholesterol was selectively accumulated in A1 bodies, and this depended on the cholesterol efflux activity of ABCA1. Treatment with either lactacystin or acetylated LDL, which reduces proteasome activity, resulted in internalization of ABCA1 in mouse peritoneal macrophages. By performing array analysis on macrophages treated with these reagents, we identified Rab4 as a key protein involved in the internalization and aberrant accumulation of ABCA1 in HEK cells. Treatment of the cells with proteasome inhibitors inhibited the degradation of Rab4, and Rab4 over-expression induced the formation of small A1 bodies. Furthermore, A1 bodies formation was substantially inhibited by silencing of the endogenous Rab4 gene. Taken together, our findings suggest that the endocytic ABCA1 possesses cholesterol efflux activity, and thus the cellular control of post-endocytic sorting, retention or recycling of functional ABCA1 in the endocytic vesicles, which is in part regulated by Rab4, is important for cholesterol metabolism in living cells.

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14 Previously, we have reported that the defect in HDL assembly in two point mutants of ABCA1 (R587W, Q597R) responsible for familial HDL deficiency is due to the impaired localization to the plasma membrane (Tanaka et al. 2003). Login to comment

[hide] ATP-binding cassette A1-mediated lipidation of apo... J Biol Chem. 2008 Jun 6;283(23):16178-86. Epub 2008 Apr 1. Denis M, Landry YD, Zha X
ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments.
J Biol Chem. 2008 Jun 6;283(23):16178-86. Epub 2008 Apr 1., [PMID:18385134]

Abstract [show]
ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that approximately 5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to approximately 1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.

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55 Primary human skin fibroblasts of normal and Tangier disease patients (Q597R) were provided by Dr. Genest (McGill University, Montreal, Canada). 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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0 The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress Arowu R. Tanaka a , Fumi Kano a , Kazumitsu Ueda b , Masayuki Murata a,* a Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan b Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan a r t i c l e i n f o Article history: Received 4 March 2008 Available online 14 March 2008 Keywords: ABCA1 CFTR ABC protein Tangier disease Cholesterol COPII ER stress ATF6 Thapsigargin DTT a b s t r a c t Mutations in ATP binding cassette transporter 1 (ABCA1), a membrane protein associated with cellular cholesterol efflux, cause Tangier disease (TD). Login to comment
1 Previously, we showed that an ABCA1 Q597R mutant (QR) identified in TD is retained in the endoplasmic reticulum. Login to comment
17 We previously found that the Q597R (QR) mutation in ABCA1, which has been identified in cases of TD, disrupts both the trafficking of ABCA1 from the ER and its ability to facilitate apoA-I induced cholesterol efflux [17]. Login to comment
25 The anti-ABCA1 antibody and the expression vectors encoding wild-type (WT) ABCA1 or the Q597R mutant, each fused with GFP, were generated as described previously [17]. Login to comment
30 HEK293 cells stably expressing WT or Q597R mutant ABCA1-GFP (HEK-WT or HEK-QR) were selected and maintained in Dulbecco`s Modified Eagle`s Medium (DMEM) supplemented with 10% fetal calf 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. Login to comment
49 Results Thapsigargin rescues the ABCA1-QR trafficking defect, but not its cholesterol efflux activity We have previously compared the intracellular localization of the wild-type ABCA1-GFP fusion protein with that of the Q597R mutant fused to GFP. Login to comment
102 (A) Fluorescence microscopy of cells transfected with WT ABCA1-GFP and ABCA1-Q597R-GFP. Login to comment
103 ABCA1-Q597R-GFP is retained in the ER. 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
101 (A) Fluorescence microscopy of cells transfected with WT ABCA1-GFP and ABCA1-Q597R-GFP. 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] Quantitative analysis of ABCA1-dependent compartme... J Biol Chem. 2008 Apr 25;283(17):11164-75. Epub 2008 Jan 24. Hassan HH, Bailey D, Lee DY, Iatan I, Hafiane A, Ruel I, Krimbou L, Genest J
Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis.
J Biol Chem. 2008 Apr 25;283(17):11164-75. Epub 2008 Jan 24., [PMID:18218626]

Abstract [show]
The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that (125)I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Delta(187-243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of (125)I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of (125)I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 degrees C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.

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7 Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). Login to comment
35 EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment
71 Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008•VOLUME 283•NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment
87 To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment
88 As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion A B 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion C ontrol Excess apoA -I -22O H /9C R A A B C A 1(Q 597R ) 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribution (ng/µgcellprotein) Hsp-70 ICCPM Tubulin 150- 70- KDa 50- C Hsp-70 ICCPM Tubulin 150- 70- KDa 50- Hsp-70 ICCPM Tubulin 150- 70- KDa 50- VLA-2 FIGURE 1. Login to comment
94 B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 ␮g/ml 125 I-apoA-I for 45 min at 37 °C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment
97 We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment
174 Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment
175 The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment
199 In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I)E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) RatioofEndocytosedABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) RatioofEndocytosedABCA1/Intα4 FIGURE 5. Login to comment
202 Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment
36 EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment
73 Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008ߦVOLUME 283ߦNUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 at SEMMELWEIS UNIV OF MEDICINE on December 3, Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment
90 To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment
91 As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0 . Login to comment
101 B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 òe;g/ml 125 I-apoA-I for 45 min at 37 &#b0;C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment
104 We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment
182 Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment
183 The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. Login to comment
184 A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment
208 In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I) E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) Ratio of Endocytosed ABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) Ratio of Endocytosed ABCA1/Int b1; 4 FIGURE 5. Login to comment
211 Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment

[hide] Identification of an ABCA1-dependent phospholipid-... J Lipid Res. 2007 Nov;48(11):2428-42. Epub 2007 Jul 26. Hassan HH, Denis M, Lee DY, Iatan I, Nyholt D, Ruel I, Krimbou L, Genest J
Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.
J Lipid Res. 2007 Nov;48(11):2428-42. Epub 2007 Jul 26., [PMID:17656736]

Abstract [show]
It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

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4 Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I- apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Login to comment
35 MATERIALS AND METHODS Patient selection For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene), as described previously (14). Login to comment
103 To test the specificity of the cross-liking and immunoprecipitation methods, the presence of a 30-fold excess of unlabeled apoA-I during the incubation of 125 I-apoA-I with 22OH/9CRA-stimulated fibroblasts, the absence of 22OH/9CRA treatment, or ABCA1 mutant fibroblasts (Q597R) drastically reduced the larger fraction of 125 I- apoA-I nonassociated with ABCA1 (Fig. 1B). Login to comment
106 The observation that the presence of ABCA1 mutant Q597R abolished the association of 125 I-apoA-I to both ABCA1 associated and nonassociated Fig. 1. Login to comment
108 A: Confluent unstimulated, 22-(R)-hydroxycholesterol/9-cis-retinoic acid (22OH/9CRA)-stimulated fibroblasts and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I in the presence or absence of a 30-fold excess of unlabeled apoA-I (+ Excess) for 45 min at 37jC. Login to comment
134 Affinity binding of apoA-I to the putative non-ABCA1 binding site To better characterize the new binding site for apoA-I, intact normal fibroblasts treated or not with 22OH/9CRA or ABCA1 mutant Q597R were incubated with increasing concentrations of 125I-apoA-I for 45 min at 37jC. Login to comment
137 At the same time, ABCA1 mutant fibroblasts (Q597R) showed no significant binding to both sites (Fig. 3D). Login to comment
159 Stimulated intact normal or ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
162 Furthermore, no significant apoA-I binding was detected in both PM and ICC fractions in ABCA1 mutant Q597R, used as a negative control in the present experiment (Fig. 5A). Login to comment
185 Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment
215 In parallel experiments, we documented that apoA-I-mediated cholesterol efflux, as well as the formation of nascent LpA-I particles, after phospholipase treatments were absent in ABCA1 mutant (Q597R) fibroblasts under conditions similar to those used in the present experiments (data not shown). Login to comment
216 On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment
227 This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment
244 A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
265 For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment
319 Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. Login to comment
186 Confluent normal (A-C) and ABCA1 mutant (Q597R) (D) fibroblasts were incubated with increasing concentrations of 125 I-apoA-I for 45 min at 37jC in the presence (A, B, D) or absence (C) of 22OH/9CRA or 300 mM glyburide (B). Login to comment
217 On the other hand, there is no evidence for lipid-free 125 I-apoA-I aggregation, as assessed by the complete removal of lipid-free 125 I-apoA-I incubated with ABCA1 mutant (Q597R) fibroblasts, under treatment or not with phospholipases by filtration with a 50,000 molecular weight cut off filter system (23). Login to comment
228 This is consistent with the results showing that overexpressing ABCA1 in BHK cells promoted the formation of the HCBS, whereas the inhibition of ABCA1 activity by glyburide or the presence of ABCA1 mutant fibroblasts (Q597R) drastically reduced the binding of apoA-I to the HCBS (Figs. 2, 3). Login to comment
245 A: Confluent stimulated normal and ABCA1 mutant (Q597R) fibroblasts were incubated with 10 mg/ml 125 I-apoA-I for 45 min at 37jC. Login to comment
266 For example: 1) inhibition of ABCA1 activity by glyburide decreased the binding of apoA-I to both ABCA1 and the HCBS; 2) rLpA-I exhibited reduced affinity for ABCA1 and also for the HCBS (Fig. 4A); and 3) ABCA1 mutants (Q597R and C1447R) that failed to bind apoA-I also failed to mediate the formation of the HCBS. Login to comment
320 Furthermore, the presence of ABCA1 mutant Q597R abolished the association of apoA-I with both compartments, consistent with the idea that ABCA1 seems to be required for the association of apoA-I with both the PM and ICCs. 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

[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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46 Indeed, patients heterozygous for the mutations R587W, Q597R, ⌬L693, N935S, A1046D, C1477R, and R2081W had between 47% and 69% of HDL-C levels of controls (Table). Login to comment
48 Six mutants, R587W, Q597R, ⌬L693, N935S, N1800H, and R2081W, showed no localization at the plasma membrane and instead accumulated intracellularly (Figure 2A), indicating that these mutations severely affect ABCA1 function by preventing its migration to the plasma membrane, thus diminishing its ability to efflux lipids and generate HDL. Login to comment
54 Of the mutants not localizing to the plasma membrane, R587W, Q597R, ⌬L693, and N935H are all EndoH sensitive, indicating that they do not exit the ER. Login to comment
58 R587W, Q597R, ⌬L693, S1506L, and R2081W showed significantly reduced cell surface ABCA1 expression, confirming our previous localization data. Login to comment
68 All 6 mutants showing no plasma membrane localization elicited significantly reduced cell surface ApoA-I binding (R587W, 33.0Ϯ8.9%, nϭ3, Pϭ0.006; Q597R, 17.4Ϯ14.0%, nϭ3, Pϭ0.009; ⌬L693, 32.6Ϯ10.6%, nϭ3, Pϭ0.008; N935S, 26.4Ϯ37.5%, nϭ3, Pϭ0.01; N1800H, 36.9Ϯ15.5%, nϭ3, Pϭ0.01; R2081W, 34.6Ϯ16.6%, nϭ3, Pϭ0.02) (Figure 3A), confirming that cell surface localization of ABCA1 is essential to elicit ApoA-I binding. Login to comment
78 Wild-type ABCA1 was localized intracellularly and at the plasma membrane. R587W, Q597R, ⌬L693, N935S, N1800H, and R2081W were only localized intracellularly. Login to comment
81 Wt ABCA1 shows both EndoH resistant and sensitive bands, indicating localization at the ER and plasma membrane. R587W, Q597R, ⌬L693, and N935S show only the lower EndoH sensitive band, indicating ER retention. 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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121 The first took advantage of the Q597R and ⌬L693 mutations, previously shown to be massively retained in the endoplasmic reticulum (3), whereas the second set comprised mutants that, although correctly expressed and trafficked to the plasma membrane, showed reduced ability to elicit the functional phenotypes associated with ABCA1 expression in transfected cells. Login to comment
122 The analysis of lysates from cells transfected with EGFP-tagged Q597R or ⌬L693 mutants either alone or in combination with wild type ungrafted ABCA1 transporter indicated that three molecular species of dimers are detectable (Fig. 3A). Login to comment
176 The mixtures contained equimolar amounts of ungrafted ABCA1 and of either Q597R or ⌬L693 variants grafted with EGFP (Q597RG or ⌬L693G). Login to comment
177 For comparison, the banding profile of ungrafted (A1WT) and EYFP-grafted (A1Y) ABCA1 is shown. Detection was performed as indicated by either the 891.3 or the anti-GFP mAb. MW, molecular weight markers. B and C, confocal analysis of cells transfected with equimolar mixtures of wild type ABCA1 grafted with ECFP (A1C) and either Q597R or ⌬L693 grafted with EGFP indicate that the presence of wild type transporter in the dimers fails to rescue the trafficking defect of both variants. Login to comment
120 The first took advantage of the Q597R and èc;L693 mutations, previously shown to be massively retained in the endoplasmic reticulum (3), whereas the second set comprised mutants that, although correctly expressed and trafficked to the plasma membrane, showed reduced ability to elicit the functional phenotypes associated with ABCA1 expression in transfected cells. Login to comment
175 The mixtures contained equimolar amounts of ungrafted ABCA1 and of either Q597R or èc;L693 variants grafted with EGFP (Q597RG or èc;L693G). Login to comment

[hide] New insights into the biogenesis of human high-den... Curr Opin Lipidol. 2006 Jun;17(3):258-67. Krimbou L, Marcil M, Genest J
New insights into the biogenesis of human high-density lipoproteins.
Curr Opin Lipidol. 2006 Jun;17(3):258-67., [PMID:16680030]

Abstract [show]
PURPOSE OF REVIEW: The interest for the human HDL system was recently revived by the identification of the ABCA1 as a critical component in the formation and maintenance of plasma HDL levels. The present review focuses on recent progress in our understanding of the basic mechanisms underlying HDL biogenesis pathways. RECENT FINDINGS: Several novel mechanisms governing ABCA1/apoA-I interactions have recently been identified: apolipoprotein A-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway; the majority of ABCA1 exists as a tetramer in human living cell, supporting the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit for the formation of nascent HDL particles; apolipoprotein A-I has been shown to have a recycling retroendocytic pathway with uptake and resecretion of the lipidated nascent HDL particles by the cell, most likely through the ABCA1 transporter pathway; there is evidence that the speciation of nascent HDL into pre-beta and alpha-HDL is linked to specific cell lines, and occurs by both ABCA1-dependent and independent pathways. SUMMARY: The fundamental mechanisms underlying the biogenesis, speciation and maturation of HDL remain complex and not well understood. Understanding the mechanisms governing HDL genesis at the cellular level could provide novel insights into the human atheroprotective system in health and disease.

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63 We have previously suggested that the high content in phosphatidylinositol [41] or the high number of apoA-I molecules per particle [43] (Fig. 2b, lower panels) may contribute to the increase in the net negative charge of nascent LpA-I and consequently cause 260 Lipid metabolism Table 1 Formation and speciation of nascent apolipoprotein (apo)A-I-containing particles in various cell lines Cell type Pre-b1-LpA-I a-LpA-I Macrophages monocyte-derived þ þ THP-1 þ þ Hepatocytes HepG2 (apoA-I endo, exo) þ þ primary mouse (apoA-I endo) þ þ CaCo-2 (apoA-I endo, exo) - þ Fibroblasts normal - þ Tangier (Q597R) - - Tangier (C1477R) - - HUVEC - - CHO - þ CHO overexpressing ABCA1 - þ The presence of pre-b1-LpA-I and a-LpA-I generated by different cell lines was determined by two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis based on our previous study [28]. 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

[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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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

[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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412 Some studies have suggested that ABCA1 proteins with substitution mutations Q597R and R587W in the first extracellular loop do not localize to the plasma membrane (232, 263), but other studies have contradicted these findings (73, 150). Login to comment
411 Some studies have suggested that ABCA1 proteins with substitution mutations Q597R and R587W in the first extracellular loop do not localize to the plasma membrane (232, 263), but other studies have contradicted these findings (73, 150). Login to comment

[hide] Biogenesis and speciation of nascent apoA-I-contai... J Lipid Res. 2005 Aug;46(8):1668-77. Epub 2005 May 16. Krimbou L, Hajj Hassan H, Blain S, Rashid S, Denis M, Marcil M, Genest J
Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines.
J Lipid Res. 2005 Aug;46(8):1668-77. Epub 2005 May 16., [PMID:15897603]

Abstract [show]
It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

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2 Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣-nascent apolipoprotein A-I-containing particles (␣-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment
26 MATERIALS AND METHODS Patient selection For the present study, we selected fibroblasts from three normal control subjects and two patients with TD (TD1, homozygous for Q597R at the ABCA1 gene; and TD2, compound heterozygous carrying the mutations C1477R and the splice site G→C in exon 24), as described previously (13, 14). Login to comment
75 However, the incomplete removal of lipid-free apoA-I from the medium may result in an artifactual presence of prebeta1-LpA-I. To ensure the quality of the complete removal of lipid-free apoA-I, the filtration of the medium from either HepG2 cells or macrophages found to form prebeta1-LpA-I was carefully controlled for each experiment by the filtration of the medium from normal fibroblasts, ABCA1 mutant Q597R fibroblasts, or lipid-free 125I-apoA-I incubated without cells. Login to comment
78 As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 ␮g/ml exogenously added 125I-apoA-I for 6 h at 37ЊC followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only ␣-LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment
83 Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller ␣-LpA-I having a diameter of ‫8ف‬ nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment
101 Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 2.5 ␮g/ml 22(R)-hydroxycholesterol and 10 ␮M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 ␮g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment
105 beled with [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and then incubated with 10 ␮g/ml lipid-free apoA-I for 24 h at 37ЊC. Radiolabeled ABCA1 mutant Q597R cells were used as a control in the present experiment. Login to comment
107 At the same time, we have not been able to detect any ␣-LpA-I formation during incubation with ABCA1 mutant Q597R (Fig. 4). Login to comment
128 In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment
151 This is also strongly supported by our results showing that glyburide did not affect the formation of prebeta1-LpA-I but had a dramatic effect on ␣-LpA-I, providing strong support for the Fig. 4. Characterization of lipidated apoA-I-containing particles generated during lipid-free apoA-I incubation with normal fibroblasts and ABCA1 mutant (Q597R). Login to comment
152 Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 ␮g/ml apoA-I for 24 h. Media were recovered and concentrated with a size-exclusion filter with a MWCO of 10,000 (without the removal of lipid-free apoA-I). Login to comment
1 Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only -nascent apolipoprotein A-I-containing particles (-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Login to comment
77 In separate experiments (see below), we show that both the centrifugal filter and the dialysis membrane retained native plasma pre1-LpA-I having an apparent molecular mass of 67 kDa. As shown in Fig. 1 (upper panels), incubation of stimulated normal fibroblasts, CaCo-2 cells, or CHO-overexpressing ABCA1 (CHO-ABCA1) with 10 g/ml exogenously added 125I-apoA-I for 6 h at 37C followed by the removal of lipid-free apoA-I as described above, and then separation of samples by 2D-PAGGE, generated only -LpA-I with a particle size ranging from 8 to 20 nm, whereas lipid-free apoA-I incubated with either HUVEC or ABCA1 mutant (Q597R) was unable to form such particles. Login to comment
82 Furthermore, inactivation of ABCA1 with glyburide in fibroblasts, CaCo-2, or CHO-ABCA1 led to the formation of smaller -LpA-I having a diameter of 8 nm; these particles were not formed in the presence of ABCA1 mutant Q597R cells. Login to comment
100 Normal fibroblasts, CaCo-2, human umbilical vein endothelial cells (HUVEC), HepG2, or ABCA1 mutant (Q597R) cells in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 2.5 g/ml 22(R)-hydroxycholesterol and 10 M 9-cis-retinoic acid (22OH/9CRA) for 20 h. THP-1 cells and human monocyte-derived macrophages in 100 mm diameter dishes were loaded with 20 g/ml cholesterol for 24 h and then stimulated with 0.3 mM cAMP for 12 h. CHO-overexpressing ABCA1 cells (CHO-ABCA1) were used as a control. Login to comment
130 Here, we present evidence that incubation of exogenously added lipid-free apoA-I with various cell lines, including human fibroblasts, CaCo-2, and CHO-ABCA1, generates only -LpA-I. In contrast, lipid-free apoA-I was unable to form such particles in the presence of HUVEC and ABCA1 mutant Q597R cells (Fig. 1), consistent with previous studies showing that lipid-free apoA-I did not promote cholesterol efflux from endothelial cells in which the expression of ABCA1 protein is very low and seems not to be sensitive to treatment with cholesterol or 22-hydroxycholesterol (24, 28). Login to comment
153 Normal fibroblasts or ABCA1 mutant (Q597R) were labeled with either [14C]cholesterol or [32P]orthophosphate as described in Materials and Methods and incubated with 10 g/ml apoA-I for 24 h. Login to comment

[hide] Structural modification of plasma HDL by phospholi... J Lipid Res. 2005 Jul;46(7):1457-65. Epub 2005 Jan 16. Hajj Hassan H, Blain S, Boucher B, Denis M, Krimbou L, Genest J
Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release.
J Lipid Res. 2005 Jul;46(7):1457-65. Epub 2005 Jan 16., [PMID:15654121]

Abstract [show]
It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

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122 Furthermore, we have previously documented that the specific binding of 125I-apoA-I to unstimulated fibroblasts was very low and totally absent in ABCA1 mutant (Q597R) fibroblasts (18). Login to comment
123 Furthermore, we have previously documented that the specific binding of 125I-apoA-I to unstimulated fibroblasts was very low and totally absent in ABCA1 mutant (Q597R) fibroblasts (18). 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
91 When these three TD mutations were introduced into ECD1 of ABCA1-GFP and the mutants were transiently or stably expressed in HEK293, R587W and Q597R appeared to be distributed mainly in the ER and not the plasma membrane. Login to comment

[hide] Effects of oxidized low density lipoprotein on the... J Huazhong Univ Sci Technolog Med Sci. 2005;25(2):113-6. Li Y, Bi H, Wu F, Zong Y, Wang Y, Qu S
Effects of oxidized low density lipoprotein on the expression and function of ABCA1 in macrophages.
J Huazhong Univ Sci Technolog Med Sci. 2005;25(2):113-6., [PMID:16116948]

Abstract [show]
In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65% cholesterol loaded by acetyl LDL (ac-LDL), and 9.78% cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with -100 microg /mL ac-LDL or with 100 microg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

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102 The gene examination of Tangier patients showed that the gene mutation of R587W and Q597R could result in the localized dysfunction of ABCA1, accumulation of cholesterol in macrophages, and the occurrence of AS. Login to comment

[hide] Characterization of oligomeric human ATP binding c... J Biol Chem. 2004 Oct 1;279(40):41529-36. Epub 2004 Jul 26. Denis M, Haidar B, Marcil M, Bouvier M, Krimbou L, Genest J
Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles.
J Biol Chem. 2004 Oct 1;279(40):41529-36. Epub 2004 Jul 26., [PMID:15280376]

Abstract [show]
The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-I-containing particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I with diameters between 9.5 and 20 nm. Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. These results demonstrate that: 1) ABCA1 exists as an oligomeric complex; and 2) ABCA1 oligomerization was independent of apoA-I binding and lipid molecules. The findings that the majority of ABCA1 exists as a tetramer that binds apoA-I, together with the observation that LpA-I contains at least four molecules of apoA-I per particle, support the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit required for the biogenesis of high density lipoprotein particles.

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9 Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment
39 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment
61 Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C with either stimulated normal or Q597R cells. Login to comment
63 15 ␮g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 ␮g/␮l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 ␮l of Me2SO to a final concentration of 1 ␮g/␮l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment
114 To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 ␮g/ml 125 I- apoA-I in 6 ml of DMEM for 24 h at 37 °C. Login to comment
123 In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment
150 Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment
152 In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment
155 Upper panels, 125 I-apoA-I (10 ␮g/ml) was incubated in DMEM for 24 h at 37 °C without cells (first gel) or with both stimulated normal and Q597R cells (second and third gels, respectively) for 24 h at 37 °C. Login to comment
159 Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment
180 We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment
199 Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment
200 These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment
8 Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. Login to comment
37 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene). Login to comment
58 The presence of labeled 125 I-apoA-I/ABCA1 complexes was directly detected by autoradiography by using XAR-2 Kodak film. Isolation of Nascent LpA-I Particles and Cross-linking with DSP- 125 I-apoA-I (10 òe;g/ml) was incubated in DMEM for 24 h at 37 &#b0;C with either stimulated normal or Q597R cells. Login to comment
60 15 òe;g of isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells was adjusted to 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, with a final protein concentration of 1 òe;g/òe;l. Immediately before an experiment, 1 mg of DSP was dissolved in 1000 òe;l of Me2SO to a final concentration of 1 òe;g/òe;l. The dissolved DSP was added to the reaction mixture for 10 DSP to 1 apoA-I molar ratio on ice. Login to comment
110 To determine the relationship between oligomeric ABCA1 complex and the structural properties of nascent apoA-I-containing particles in our cell culture model, stimulated cells either from normal or from Tangier disease (Q597R) subjects in 100-mm diameter dishes were incubated with 10 òe;g/ml 125 I-apoA-I in 6 ml of DMEM for 24 h at 37 &#b0;C. Login to comment
120 In contrast, lipid-free apoA-I incubated with stimulated ABCA1 mutant (Q597R) cells was unable to form such particles (third gel), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (first gel). Login to comment
145 Either lipid-free apoA-I incubated without cells or lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells were used as controls. Login to comment
147 In contrast, both lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells and lipid-free apoA-I incubated without cells remained in the monomeric form following cross-linking. Login to comment
154 Lower panel, isolated LpA-I generated by normal cells, apoA-I incubated with Q597R cells, or lipid-free apoA-I incubated without cells were treated with DSP as described under "Experimental Procedures." Login to comment
175 We demonstrate that lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells remained in the monomeric form (Fig. 5, lower panel). Login to comment
194 Although ABCA1 mutants Q597R and C1477R were found to oligomerize normally (data not shown) and localized to the plasma membrane, they showed the total absence of binding to apoA-I (21, 23). Login to comment
195 These results indicate that the apoA-I lipidation defect observed in either Q597R or C1477R ABCA1 mutants is not caused by impaired oligomerization of ABCA1. Login to comment

[hide] Molecular and cellular physiology of apolipoprotei... J Biol Chem. 2004 Feb 27;279(9):7384-94. Epub 2003 Dec 4. Denis M, Haidar B, Marcil M, Bouvier M, Krimbou L, Genest J Jr
Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).
J Biol Chem. 2004 Feb 27;279(9):7384-94. Epub 2003 Dec 4., [PMID:14660648]

Abstract [show]
The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) = 1.4 +/- 0.66 h; n = 3) but almost completely inhibited at either 15 or 4 degrees C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited alpha-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated alpha-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates alpha-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.

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6 A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. Login to comment
38 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment
62 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
126 In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment
129 Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment
130 As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment
139 In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment
140 In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 ␮g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 °C. Login to comment
143 In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment
196 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
197 B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 ␮g/ml of 125 I-apoA-I for 1 h at 37 °C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 ␮g/ml). Login to comment
233 B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 ␮g/ml of apoA-I or 1 mg/ml of BSA at 37 °C for the indicated time points. Login to comment
242 125 I- apoA-I was incubated in DMEM/BSA for 24 h at 37 °C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 °C. Login to comment
253 to form larger particles during its incubation with Q597R mutant cells. Login to comment
37 EXPERIMENTAL PROCEDURES Patient Selection-For the present study, we selected fibroblasts from 3 normal control subjects and 1 patient with TD (homozygous for Q597R at the ABCA1 gene). Login to comment
61 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
124 In addition, ABCA1 mutant (Q597R) that has been shown previously to not cross-link to apoA-I (13) was used as a negative control for the present experiment and showed no binding or cross-linking to ABCA1 (Fig. 3, A and B). Login to comment
127 Also, to rule out the possibility that treatment with phospholipases might induce membrane aggregation or affect ABCA1 protein structure, which may trap apoA-I and result in nonspecific cross-linking, we examined the effect of phospholipases treatment on the cross-linking of 125 I-apoA-I to ABCA1 mutant (Q597R). Login to comment
128 As shown in Fig. 3B (upper panel), 125 I-apoA-I did not cross-link to Q597R mutant whether treated with PC-PLC, SM-ase, or left intact. Login to comment
137 In addition, to ensure that cholesterol efflux was ABCA1-dependent in our cell culture system, apoA-I-mediated cholesterol efflux from ABCA1 mutant (Q597R) cells was also monitored. Login to comment
138 In order to investigate the nature of apoA-I-containing particles generated by ABCA1 activity, stimulated cells from either normal or from TD (Q597R) subjects in 100 mm diameter dishes were incubated with 10 òe;g/ml of 125 I-apoA-I in 8 ml of DMEM for 24 h at 37 &#b0;C. Login to comment
141 In contrast, lipid-free apoA-I incubated with stimulated mutant Q597R cells was unable to form such particles (panel C), which had a molecular diameter and charge similar to the lipid-free apoA-I incubated in the same conditions without cells (panel A). Login to comment
194 ABCA1 mutant (Q597R) was used as a negative control. Login to comment
195 B, upper panel, intact stimulated normal or Q597R cells in 100-mm diameter dishes were incubated or not with PC-PLC or SM-ase as described above and then incubated with 10 òe;g/ml of 125 I-apoA-I for 1 h at 37 &#b0;C in the presence or absence of a 20-fold excess of unlabeled apoA-I (200 òe;g/ml). Login to comment
230 B, stimulated normal or Q597R cells were radiolabeled with [3 H]cholesterol and incubated with 10 òe;g/ml of apoA-I or 1 mg/ml of BSA at 37 &#b0;C for the indicated time points. Login to comment
239 125 I-apoA-I was incubated in DMEM/BSA for 24 h at 37 &#b0;C without cells (A) or with both stimulated normal and Q597R cells (B and C, respectively) for 24 h at 37 &#b0;C. Login to comment
249 Results shown are representative of two different independent experiments. Lipidation of ApoA-I by ABCA1 to form larger particles during its incubation with Q597R mutant cells. Login to comment

[hide] Familial HDL deficiency due to ABCA1 gene mutation... Atherosclerosis. 2004 Feb;172(2):309-20. Pisciotta L, Hamilton-Craig I, Tarugi P, Bellocchio A, Fasano T, Alessandrini P, Bon GB, Siepi D, Mannarino E, Cattin L, Averna M, Cefalu AB, Cantafora A, Calandra S, Bertolini S
Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders.
Atherosclerosis. 2004 Feb;172(2):309-20., [PMID:15019541]

Abstract [show]
Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors.

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204 The E284K and the Y482C are located in the first extracellular loop of ABCA1, where they may interfere with the binding to Apo A-I and/or the membrane release of phospholipids, as it has been demonstrated for other mutations (R587W and Q597R) located in the same extracellular domain [40,41]. Login to comment
203 The E284K and the Y482C are located in the first extracellular loop of ABCA1, where they may interfere with the binding to Apo A-I and/or the membrane release of phospholipids, as it has been demonstrated for other mutations (R587W and Q597R) located in the same extracellular domain [40,41]. 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] 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
79 This is indeed the case with mutant Q597R,58,60 which shows no ApoA-I binding. 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
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
71 This is indeed the case with mutant Q597R,58,60 which shows no ApoA-I binding. 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] 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] Cellular phospholipid and cholesterol efflux in hi... Circulation. 2003 Mar 18;107(10):1366-71. Marcil M, Bissonnette R, Vincent J, Krimbou L, Genest J
Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency.
Circulation. 2003 Mar 18;107(10):1366-71., [PMID:12642355]

Abstract [show]
BACKGROUND: Prospective studies have examined the relationship between coronary artery disease and low plasma levels of high-density lipoprotein cholesterol (HDL-C). METHODS AND RESULTS: We investigated the causes of hypoalphalipoproteinemia (HypoA; HDL-C <5th percentile) in 64 subjects (12 women and 52 men). Apolipoprotein AI-mediated cellular cholesterol and phospholipid efflux were measured in fibroblasts from HypoA subjects, 9 controls, 2 patients with Tangier disease, and 5 patients with hyperalphalipoproteinemia. A phospholipid efflux defect was defined as <70% of controls. Mean HDL-C was 0.49+/-0.21 mmol/L. Cholesterol and phospholipid efflux correlated strongly (r=0.72, P<0.001). Phospholipid efflux and HDL-C (r=0.64, P<0.001) correlated in HypoA subjects. However, phospholipid or cholesterol efflux was no longer a determinant of HDL-C levels at higher levels (> approximately 1.0 mmol/L) of HDL-C. In HypoA subjects, 4 cases of Tangier disease and 6 of familial HDL deficiency (heterozygous Tangier disease) were identified (10 of 64; 16%). In the remaining 54 subjects, mean lipid efflux was not significantly different from controls and subjects with hyperalphalipoproteinemia. A phospholipid efflux defect was identified in 7 additional HypoA subjects, and a cholesterol efflux defect was detected in 11 subjects. In 2 of these subjects, the ABCA1 gene was ruled out as the cause of the efflux defect, while in 3, the low HDL-C trait segregated with the ABCA1 gene locus. CONCLUSIONS: Lipidation of lipid-poor apolipoprotein AI may not be a major determinant of cholesterol accumulation within more mature HDL particles and increasing cholesterol or phospholipid efflux beyond normal levels may not lead to increase in plasma HDL-C levels. ABCA1 is essential in the initial steps of HDL formation but other plasma events are major modulators of HDL-C levels.

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85 Molecular Characterization of ABCA1 Gene in Study Subjects Cell Lines HDL-C, mmol/L Nucleotide Change Predicted Protein Alteration TD CTL-1 0.10 Exon 30 T4369C; exon 24 splice site G3C C1477R; part of the transcript deleted TD CTL-2 0.15 Exon 13 A1730G Q597R FHD-1 0.40 Exon 14 ⌬2017-9 ⌬L693 FHD-2 0.18 Exon 48 C6370T R2144X FHD-3 0.39 Exon 41 ⌬5618-23 ⌬ED1893,4 FHD-4 0.18 Exon 18 C2665T R909X FHD-5 0.10 Exon 23 T3667C M1091T FHD-6 0.57 Exon 49 C6844T P2150L, R587W TD-1 0.03 Exon 48 ⌬C6370; ND 2145X TD-2 0.07 ND ND TD-3 0.03 ND 2203X TD-4 0.09 Exon 19 C3181T; ND T929I; ND CTL indicates control. Login to comment
79 Molecular Characterization of ABCA1 Gene in Study Subjects Cell Lines HDL-C, mmol/L Nucleotide Change Predicted Protein Alteration TD CTL-1 0.10 Exon 30 T4369C; exon 24 splice site G3C C1477R; part of the transcript deleted TD CTL-2 0.15 Exon 13 A1730G Q597R FHD-1 0.40 Exon 14 èc;2017-9 èc;L693 FHD-2 0.18 Exon 48 C6370T R2144X FHD-3 0.39 Exon 41 èc;5618-23 èc;ED1893,4 FHD-4 0.18 Exon 18 C2665T R909X FHD-5 0.10 Exon 23 T3667C M1091T FHD-6 0.57 Exon 49 C6844T P2150L, R587W TD-1 0.03 Exon 48 èc;C6370; ND 2145X TD-2 0.07 ND ND TD-3 0.03 ND 2203X TD-4 0.09 Exon 19 C3181T; ND T929I; ND CTL indicates control. 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
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
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
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
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
74 ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. 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
84 The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. Login to comment
85 The Q597R mutant almost completely lost this activity. 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
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
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
125 R587W and Q597R ABCA1-GFP appeared to be retained in the ER. 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
128 Fitzgerald et al. (17) reported that R587W or Q597R mutation did not affect the PM localization but disrupted the direct interaction with ApoA-I. This supports a major conformational alteration of ECD1 by these mutations. 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
67 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
73 ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. 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
118 ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. Login to comment
124 R587W and Q597R ABCA1-GFP appeared to be retained in the ER. Login to comment

[hide] cAMP induces ABCA1 phosphorylation activity and pr... J Lipid Res. 2002 Dec;43(12):2087-94. Haidar B, Denis M, Krimbou L, Marcil M, Genest J Jr
cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts.
J Lipid Res. 2002 Dec;43(12):2087-94., [PMID:12454270]

Abstract [show]
ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.

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176 Molecular characterization of ABCA1 gene in study subjects Cell Lines HDL-C Nucleotide Change Predicted Protein Alteration mmol/l CTR1 1.63 - - CTR2 1.20 - - FHD1 0.27 Exon 14 ⌬2017-9 ⌬L693 FHD2 0.18 Exon 18 C2665T R909X FHD3 0.39 Exon 41 ⌬5618-23 ⌬ED1893,4 FHD4 0.18 Exon 48 C6370T R2144X FHD5 0.09 Exon 36 GG5277,8C fs 1628G, Q1636X TD1 Ͻ0.1 Exon 30 T4369C; Exon 24 splice site G→C C1477R; Part of the transcript deleted TD2 Ͻ0.1 Exon 13 A1730G Q597R TD3 Ͻ0.1 Exon 48 ⌬C6370; nd 2145X; nd FHD1-5 are heterozygous for the reported mutation; TD1,3 are compound heterozygous and TD2 is homozygous. 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
153 Two of the Tangier transporters (namely Q597R and ⌬L693) reach the plasma membrane very inefficiently, and as a consequence fail to elicit any significant function as measured by surface binding of either annexin V or Fig. 4. Login to comment
161 Q597R and ⌬L693 are virtually absent from the cell surface while expressed normally. Login to comment
199 Confocal images of ABCA1 mutant alleles expressed in HeLa cells (upper panel) indicate that in the case of Q597R the mutation affects intracellular trafficking and leads to retention into the ER. 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
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
152 Two of the Tangier transporters (namely Q597R and L693) reach the plasma membrane very inefficiently, and as a consequence fail to elicit any significant function as measured by surface binding of either annexin V or Fig. 4. Login to comment
160 Q597R and L693 are virtually absent from the cell surface while expressed normally. Login to comment
198 Confocal images of ABCA1 mutant alleles expressed in HeLa cells (upper panel) indicate that in the case of Q597R the mutation affects intracellular trafficking and leads to retention into the ER. 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

[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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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
198 Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). 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
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
190 Three of the mutants (Q597R, C1477R, and S1506L) had dramatic reductions in their cross-linking efficiency to apoA-I, relative to the wild type transporter (90% or greater reduction). 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

[hide] Characterization and classification of ATP-binding... J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7. Matsumura Y, Ban N, Ueda K, Inagaki N
Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.
J Biol Chem. 2006 Nov 10;281(45):34503-14. Epub 2006 Sep 7., [PMID:16959783]

Abstract [show]
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.

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219 For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment
218 For example, R587W and Q597R mutations of ABCA1, which are found in Tangier disease patients with high density lipoprotein deficiency, appear to be impaired in intracellular trafficking and localized predominantly to the ER (36). Login to comment

[hide] Functional rescue of mutant ABCA1 proteins by sodi... J Lipid Res. 2013 Jan;54(1):55-62. doi: 10.1194/jlr.M027193. Epub 2012 Oct 20. Sorrenson B, Suetani RJ, Williams MJ, Bickley VM, George PM, Jones GT, McCormick SP
Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.
J Lipid Res. 2013 Jan;54(1):55-62. doi: 10.1194/jlr.M027193. Epub 2012 Oct 20., [PMID:23087442]

Abstract [show]
Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

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161 Plasma membrane localization of the p.Q597R protein was promoted with thapsigargin, an inducer of ER stress, whereas 4-PBA is known to alleviate ER stress (14) by acting as a chaperone itself (13) as well as inducing the expression of endogenous chaperone proteins (15). Login to comment
195 Improved positioning of ABCA1 at the plasma membrane has been shown previously for a p.Q597R mutant under conditions of induced ER stress; however, in that instance, no improvement in efflux activity was seen (23). Login to comment
196 This is in contrast with our results where the majority of the mutants showed significant improvements in function, including the p.A594T mutant, which is of a similar location to p.Q597R. 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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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