ABCMdb

ABCB1 p.Thr769Cys

Predicted by SNAP2:	A: N (66%), C: N (57%), D: N (53%), E: D (80%), F: D (71%), G: D (53%), H: D (71%), I: N (57%), K: D (85%), L: D (53%), M: D (63%), N: D (66%), P: D (75%), Q: D (75%), R: D (80%), S: N (87%), V: N (61%), W: D (80%), Y: D (75%),
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: N, V: D, W: D, Y: D,

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[hide] Disulfide cross-linking analysis shows that transm... J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10. Loo TW, Bartlett MC, Clarke DM
Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PMID:14670948]

Abstract [show]
Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.

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124 The verapamiland demecolcine-stimulated ATPase activities rel- TABLE I Cross-linking between residues in TMs 5 and 8 TM5 TM 8 F767C I768C T769C F770C F771C L772C Q773C G774C F775C T776C I293C -a - - - - - - - ϩϩb - T294C - - - - - - - - - - A295C - - - - - - - ϩ - - N296C - - - ϩϩb ϩ - ϩ ϩϩc ϩ - I297C - - - ϩd ϩϩ - - ϩϩb - - S298C - - - - - - - - - - I299C - - - ϩϩc - - - ϩϩc - - G300C ϩϩe - - ϩϩc ϩϩb - - ϩϩb ϩ - A301C - - - - - - - - - - A302C - - - - - - - - - - a No cross-linked product detected in SDS-PAGE gels at 37 °C. b Cross-linked product was also detected at 22 °C. c Cross-linked product was also detected at 22 °C and at 4 °C. d Relatively weak cross-linking (Ͻ50% of P-gp cross-linked) at 37 °C. e Relatively strong cross-linking (Ͼ50% of P-gp cross-linked) at 37 °C. FIG. 2. Login to comment

[hide] Mechanisms of azole resistance in Candida albicans... Res Microbiol. 2015 Apr;166(3):153-61. doi: 10.1016/j.resmic.2015.02.009. Epub 2015 Mar 6. Liu JY, Shi C, Wang Y, Li WJ, Zhao Y, Xiang MJ
Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China.
Res Microbiol. 2015 Apr;166(3):153-61. doi: 10.1016/j.resmic.2015.02.009. Epub 2015 Mar 6., [PMID:25748216]

Abstract [show]
This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans (n = 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GC-MS chromatogram, and no missense mutations were found in ERG3. Furthermore, seven amino acid substitutions in ERG11, A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole.

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No. Sentence Comment
103 of strainsa Site of isolation FLC ITRA VRC Amino acid substitution(s) in Tac1b Missense mutation in TAC1 gene Amino acid substitution(s) in Erg11b Missense mutation in ERG11 gene Gene overexpression Y141 Vagina 16(SDD) >16(R) 0.5(S) S199N, A736V, L935S G596A, C2207T, T2804C A114S, Y205E, Y257H, V437I G340T, T613G or C615A, T769C, G1309A CDR2 Y201 Vagina 16(SDD) 16(R) 1(S) F104V, A736V, L935S T310G, C2207T, T2804C A114S, Y205E, Y257H, V437I G340T, T613G or C615A, T769C, G1309A CDR1 Y205 Vagina 16(SDD) 0.5(SDD) 2(I) D776N, L935S G2326A, T2804C Y132H, Y205E,V437I, G448E T394C, T613G or C615A, G1309A, G1343A CDR1,CDR2 Y206 Vagina 16(SDD) 16(R) 2(I) S199N, D776N, L935S G596A, G2326A, T2804C Y132H, Y205E, V437I, G472R T394C, T613G or C615A, G1309A, G1414A CDR2 Y208 Vagina 32(SDD) 16(R) 1(S) e e Y132H, Y205E, N435V, G448E,D502E T394C, T613G or C615A, A1303G, G1343A, C1506A e Y210 Vagina 16(SDD) 0.25(SDD) 4(R) S199N, N772K, N896S G596A, T2316A, A2687G Y132H, Y205E, Y257H, E260V, V437I, G448E T394C, T613G or C615A, T769C, A779T, G1309A, G1343A CDR1,CDR2 Y203 Vagina 0.5(S) 0.0313(S) <0.0313(S) S199N, L935S G596A, T2804C Y205E, V437I T613G or C615A, G1309A e J026 Sputum 0.5(S) 0.0313(S) 0.0313(S) e e Y205E, V437I T613G or C615A, G1309A e J509 Vagina 32(SDD) 0.25(SDD) 0.25(S) S199N, D776N, L935S G596A, G2326A, T2804C D116E, K128T, Y205E, V437I T348A, A383C, T613G or C615A, G1309A CDR2 J774 Sputum 0.5(S) 0.0313(S) <0.0313(S) e e Y205E, V437I T613G or C615A, G1309A e J5039 Sputum 2(S) 0.0313(S) <0.0313(S) F104V, L935S T310G, T2804C D116E T348A e J5043 Sputum 2(S) 0.0313(S) <0.0313(S) e e Y205E, V437I T613G or C615A, G1309A e H24308 Skin 32(SDD) 0.5(SDD) 0.25(S) S199N, D776N G596A, G2326A, D116E, K128T, K143R, Y205E, V437I T348A, A383C, A428G, T613G or C615A, G1309A CDR2 H75045 Vagina 16(SDD) 0.25(SDD) 2(I) S199N, L935S, F964Y G596A, T2804C, A2890G or A2891T Y132H, Y205E, V437I, G448E T394C, T613G or C 615A, G1309A, G1343A CDR1,CDR2,ERG11 H13139 Skin >64(R) >16(R) 8(R) e e D116E, Y132F, K143Q, Y205E, Y257H T348A, A395T, A427C, T613G or C615A, T769C e H21897 Skin >64(R) >16(R) 16(R) S199N, N772K, N896S G596A, T2316A, A2687G D116E, Y132F, K143Q, Y205E, V437I T348A, A395T, A427C, T613G or C615A, G1309A CDR1,CDR2 H64110 Skin >64(R) 16(R) 4(R) e e D116E, Y132F, K143Q, Y205E, Y257H T348A, A395T, A427C, T613G or C615A, T769C e H30071 Sputum 4(S) <0.0313(S) 0.0313(S) e e D116E T348A e Rc701 Sputum 0.25(S) 0.0313(S) <0.0313(S) e e Y205E, V437I T613G or C615A, G1309A e RC837 Sputum 0.25(S) 0.0313(S) <0.0313(S) S199N, L935S G596A, T2804C D116E T348A e Rc924 Sputum 0.125(S) <0.0313(S) <0.0313(S) F104V, L935S T310G, T2804C Y205E,E226D,V437I T613G or C615A, A789C, G1309A e Rc289 Throat 0.25(S) 0.0313(S) 0.0313(S) e e Y205E, V437I T613G or C615A, G1309A e Rc273 Sputum 0.25(S) <0.0313(S) 0.0313(S) e e D116E, V437I T348A, G1309A e Rc923 Sputum 0.25(S) 0.0313(S) 0.0313(S) S199N, L935S G596A, T2804C Y205E, A255V, V437I T613G or C615A, C764T, G1309A e Rc592 Sputum 0.25(S) 0.0313(S) <0.0313(S) e e D116E, Y205E T348A, T613G or C615A e Rc271 Stool 0.25(S) <0.0313(S) <0.0313(S) F104V, L935S T310G, T2804C D116E, V437I T348A, G1309A e Rc286 Sputum 0.25(S) 0.0313(S) 0.0313(S) e Y205E, V437I T613G or C615A, G1309A e Rc944 Sputum 1(S) 0.0313(S) 0.0313(S) e D116E T348A e Rc963 Throat 0.25(S) 0.0313(S) 0.0313(S) e Y205E, V437I T613G or C615A, G1309A e Rc827 Sputum 0.125(S) <0.0313(S) <0.0313(S) e Y205E, V437I T613G or C615A, G1309A e a Y, Shanghai First Maternity and Infant Hospital at Tonji University School of Medicine. Login to comment