ABCA1 p.Val93Cys
| Predicted by SNAP2: | A: N (53%), C: N (61%), D: D (66%), E: D (63%), F: N (72%), G: D (66%), H: D (53%), I: N (87%), K: D (66%), L: N (82%), M: N (72%), N: N (53%), P: D (66%), Q: N (78%), R: D (53%), S: N (57%), T: N (66%), W: D (59%), Y: N (61%), |
| Predicted by PROVEAN: | A: N, C: N, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: N, R: D, S: N, T: N, W: D, Y: N, |
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[hide] An analysis of the role of a retroendocytosis path... J Lipid Res. 2008 Jun;49(6):1322-32. Epub 2008 Mar 22. Faulkner LE, Panagotopulos SE, Johnson JD, Woollett LA, Hui DY, Witting SR, Maiorano JN, Davidson WS
An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages.
J Lipid Res. 2008 Jun;49(6):1322-32. Epub 2008 Mar 22., [PMID:18359958]
Abstract [show]
The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.
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No. Sentence Comment
131 The V93C mutant was chosen for further studies because it exhibited the highest levels of expression in our recombinant system.
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ABCA1 p.Val93Cys 18359958:131:4
status: NEW132 1324 Journal of Lipid Research Volume 49, 2008 atHealthScienceLibraryCB#7585,onSeptember24,2012www.jlr.orgDownloadedfrom 0.DC1.html Next, the ability to visualize fluorescent apoA-I (V93C) within RAW264.7 macrophages treated with cAMP to induce expression of ABCA1 was assessed.
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ABCA1 p.Val93Cys 18359958:132:149
status: NEWX
ABCA1 p.Val93Cys 18359958:132:184
status: NEW133 Panels 1 and 2 of Fig. 2A show an optical slice taken through the center of the macrophage by confocal microscopy after incubation with fluorescent apoA-I (V93C) for 2 h.
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ABCA1 p.Val93Cys 18359958:133:156
status: NEW143 To evaluate this possibility, the effect of cAMP treatment on the uptake of dextran polymers labeled with the identical Alexa Fluor 546 probe as that present on our apoA-I (V93C) was studied.
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ABCA1 p.Val93Cys 18359958:143:173
status: NEW181 Colocalization of Alexa Fluor apoA-I (V93C) with LysoTracker Green in RAW cells.
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ABCA1 p.Val93Cys 18359958:181:38
status: NEW190 Then, either 10 mg/ml Alexa Fluor-labeled apoA-I (V93C) 6 400 mg/ml unlabeled apoA-I competitor, or 18 mg/ml Alexa Fluor-labeled dextran beads were added 6 0.3 mM cAMP for 2 h.
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ABCA1 p.Val93Cys 18359958:190:50
status: NEW224 Pulse-chase incubation of Alexa Fluor apoA-I (V93C) in RAW cells.
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ABCA1 p.Val93Cys 18359958:224:46
status: NEW223 Pulse-chase incubation of Alexa Fluor apoA-I (V93C) in RAW cells.
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ABCA1 p.Val93Cys 18359958:223:46
status: NEW[hide] Cyclosporin A traps ABCA1 at the plasma membrane a... Arterioscler Thromb Vasc Biol. 2004 Nov;24(11):2155-61. Epub 2004 Sep 9. Le Goff W, Peng DQ, Settle M, Brubaker G, Morton RE, Smith JD
Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I.
Arterioscler Thromb Vasc Biol. 2004 Nov;24(11):2155-61. Epub 2004 Sep 9., [PMID:15358601]
Abstract [show]
OBJECTIVE: ABCA1 mediates cellular cholesterol and phospholipid efflux to apolipoprotein A-I and other apolipoprotein acceptors. In this study, we analyzed the effect of the immunosuppressant cyclosporin A on the ABCA1-mediated lipid effluxes reactions. METHODS AND RESULTS: Cyclosporin A acted as a potent inhibitor of ABCA1 activity in several cell lines. Using the RAW264.7 mouse macrophage cell line, in which ABCA1 and its associated cholesterol efflux activity are inducible by cAMP analogues, cyclosporin A inhibition of cholesterol efflux to apolipoprotein A-I was rapidly reversible after its removal from the culture media, implying that ABCA1 levels were not drastically reduced by cyclosporin A. In fact, cyclosporin A treatment decreased ABCA1 turnover and yielded a 2-fold increase in cell-surface ABCA1. Despite the increase in cell-surface ABCA1, cyclosporin A decreased apolipoprotein A-I uptake, resecretion, and degradation in RAW cells. Finally, consistent with the inhibition of ABCA1 in vitro, cyclosporin A treatment induced a 33% reduction of high-density lipoprotein (HDL) levels in mice. CONCLUSIONS: ABCA1 inhibition by cyclosporin A supports a role for ABCA1 endocytic trafficking in ABCA1-mediated lipid efflux and could explain in part the low HDL levels observed in some patients with transplants.
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50 ApoAI Uptake, Degradation, and Resecretion Assay Radiolabeling of 140 g of human ApoAI (specific activity 3620 cpm/ng) and assay of [125 I]apoAI uptake, degradation, and resecretion were performed as previously described.21 Uptake of Alexa568-labeled apoAI, processing, fixation, and DAPI counterstaining were as previously described,19 with the following modifications: an N-terminal His tagged ApoAI V93C substitution was created and purified by standard methods22 and labeled with Alexa 568 Maleimide (Molecular Probes).
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ABCA1 p.Val93Cys 15358601:50:410
status: NEW44 ApoAI Uptake, Degradation, and Resecretion Assay Radiolabeling of 140 òe;g of human ApoAI (specific activity 3620 cpm/ng) and assay of [125 I]apoAI uptake, degradation, and resecretion were performed as previously described.21 Uptake of Alexa568-labeled apoAI, processing, fixation, and DAPI counterstaining were as previously described,19 with the following modifications: an N-terminal His tagged ApoAI V93C substitution was created and purified by standard methods22 and labeled with Alexa 568 Maleimide (Molecular Probes).
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ABCA1 p.Val93Cys 15358601:44:409
status: NEW