ABCG1 p.Tyr667Leu
| Predicted by SNAP2: | A: D (66%), C: D (59%), D: D (85%), E: D (85%), F: N (66%), G: D (75%), H: D (75%), I: D (63%), K: D (80%), L: D (63%), M: D (63%), N: D (80%), P: D (91%), Q: D (80%), R: D (80%), S: D (71%), T: D (66%), V: D (63%), W: D (59%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, |
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[hide] Cholesterol sensing by the ABCG1 lipid transporter... Biochim Biophys Acta. 2015 Jul;1851(7):956-64. doi: 10.1016/j.bbalip.2015.02.016. Epub 2015 Feb 27. Sharpe LJ, Rao G, Jones PM, Glancey E, Aleidi SM, George AM, Brown AJ, Gelissen IC
Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain.
Biochim Biophys Acta. 2015 Jul;1851(7):956-64. doi: 10.1016/j.bbalip.2015.02.016. Epub 2015 Feb 27., [PMID:25732853]
Abstract [show]
The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.
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No. Sentence Comment
107 Cholesterol docking studies were performed for the Y667 containing CRAC motifs as well as the Y667L ABCG1 mutant.
X
ABCG1 p.Tyr667Leu 25732853:107:94
status: NEW109 Y667 in this model was replaced by leucine to simulate effects of the Y667L mutation on the interaction between this ABCG1 CRAC motif and cholesterol.
X
ABCG1 p.Tyr667Leu 25732853:109:70
status: NEW147 Individual point mutations of Y649L, Y667L and Y672L were introduced into ABCG1 and stable cell lines overexpressing the individual mutants generated.
X
ABCG1 p.Tyr667Leu 25732853:147:37
status: NEW148 Fig. 3A shows that the activity of both the Y649L and Y667L mutants was totally abolished, while the Y672L mutant had reduced cholesterol export activity (this latter difference was not statistically significant).
X
ABCG1 p.Tyr667Leu 25732853:148:54
status: NEW149 Upon investigation of the protein stability in response to cholesterol starvation/enrichment, the Y667L mutant ABCG1 protein was not stabilised by addition of cholesterol (Fig. 3B).
X
ABCG1 p.Tyr667Leu 25732853:149:98
status: NEW150 Some experiments suggested that the degradation of the Y667L mutant upon starvation was more pronounced than the wild type, however this was not consistent.
X
ABCG1 p.Tyr667Leu 25732853:150:55
status: NEW153 Y667L is degraded via the ubiquitin-proteasomal system As mentioned, we showed previously that ABCG1 is ubiquitinated and degraded by the proteasome when cholesterol levels drop in the cell [5].
X
ABCG1 p.Tyr667Leu 25732853:153:0
status: NEW154 Investigation of the route of degradation of the Y667L mutant showed that this protein was also degraded via the ubiquitin-proteasomal system as was previously shown for the wild type ABCG1 (Fig. 3C).
X
ABCG1 p.Tyr667Leu 25732853:154:49
status: NEW155 Overexposure of the Western blot after incubation with a proteasomal inhibitor (MG132) revealed a laddering and smearing pattern, which may indicate that Y667L protein is also ubiquitinated (Fig. 3C).
X
ABCG1 p.Tyr667Leu 25732853:155:154
status: NEW156 Addition of lysosomal inhibitors, chloroquine and ammonium chloride, did not increase the Y667L protein levels, indicating that this pathway of degradation was not quantitatively important.
X
ABCG1 p.Tyr667Leu 25732853:156:90
status: NEW158 Staining patterns were the same for the wild type and Y667L mutant, displaying a largely perinuclear distribution, which is identical to what we have shown previously for these cells (Supplementary Fig. 5; [12]).
X
ABCG1 p.Tyr667Leu 25732853:158:54
status: NEW174 2A) Cholesterol export of parental CHOK1 cells (CHOK1), CHOK1 cells overexpressing wild-type ABCG1 (ABCG1), or two independent clones overexpressing triple Y649L, Y667L and Y672L mutants (Triple Y/L).
X
ABCG1 p.Tyr667Leu 25732853:174:163
status: NEW181 In view of the above, and the marked effect of the Y667L mutation on both cholesterol-mediated rescue of ABCG1, as well as cholesterol efflux, we aimed to investigate the interaction of cholesterol with the Y667 CARC motif, by docking a cholesterol molecule to the structural homology model of the area containing residues F652 to R678.
X
ABCG1 p.Tyr667Leu 25732853:181:51
status: NEW193 Docking of cholesterol to the Y667L mutant Considering that the Y667L mutant had no transport capacity and had lost its cholesterol-mediated stability, we performed an identical simulation by docking cholesterol to this mutant (Fig. 5; Y667L).
X
ABCG1 p.Tyr667Leu 25732853:193:30
status: NEWX
ABCG1 p.Tyr667Leu 25732853:193:64
status: NEWX
ABCG1 p.Tyr667Leu 25732853:193:236
status: NEW203 3A) Cholesterol export of CHOK1 cells overexpressing ABCG1 with a single mutation (Y649L, Y667L or Y672L).
X
ABCG1 p.Tyr667Leu 25732853:203:90
status: NEW205 Statistically significant differences in HDL-mediated export (via 2-way ANOVA; p b 0.05) are indicated between cells overexpressing ABCG1 vs CHOK1, Y649L and Y667L mutants.
X
ABCG1 p.Tyr667Leu 25732853:205:158
status: NEW211 3C) Left side: ABCG1 Y667L protein levels at T = 0 or after 8 h of treatment with BSA alone (0.1% in F12 media) or with addition of MG132 (20 bc;M), ammonium chloride (20 bc;'c;) or chloroquine (CQ, 200 bc;M).
X
ABCG1 p.Tyr667Leu 25732853:211:21
status: NEW225 Time course of ligand binding to the wild type and Y667L mutant ABCG1 TM6 area.
X
ABCG1 p.Tyr667Leu 25732853:225:51
status: NEW231 (Y667L) Y667L mutant.
X
ABCG1 p.Tyr667Leu 25732853:231:1
status: NEWX
ABCG1 p.Tyr667Leu 25732853:231:8
status: NEW239 This model was further supported by an identical analysis of the Y667L mutant, where the binding locus of the cholesterol was altered due to the changed position of the R671 side chain.
X
ABCG1 p.Tyr667Leu 25732853:239:65
status: NEW251 In addition to rendering ABCG1 inactive, the Y667L mutation also altered the post-translational regulation of the transporter protein by cholesterol.
X
ABCG1 p.Tyr667Leu 25732853:251:45
status: NEW253 Considering that the Y667L protein was not stabilised by addition of cholesterol, this implies that binding of cholesterol to this site is necessary for "rescue" from degradation to occur.
X
ABCG1 p.Tyr667Leu 25732853:253:21
status: NEW