ABCB1 p.Phe804Asp
| Predicted by SNAP2: | A: D (85%), C: D (80%), D: D (95%), E: D (91%), G: D (91%), H: D (71%), I: D (85%), K: D (95%), L: D (85%), M: D (71%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (75%), Y: N (82%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: 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, Y: N, |
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[hide] Tariquidar inhibits P-glycoprotein drug efflux but... Biochem Pharmacol. 2014 Dec 15;92(4):558-66. doi: 10.1016/j.bcp.2014.10.006. Epub 2014 Oct 22. Loo TW, Clarke DM
Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation.
Biochem Pharmacol. 2014 Dec 15;92(4):558-66. doi: 10.1016/j.bcp.2014.10.006. Epub 2014 Oct 22., [PMID:25456855]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is a drug pump that confers multidrug resistance. Inhibition of P-gp would improve chemotherapy. Tariquidar is a potent P-gp inhibitor but its mechanism is unknown. Here, we tested our prediction that tariquidar inhibits P-gp cycling between the open and closed states during the catalytic cycle. Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Residues A80C/R741C come close enough (<7A) to spontaneously cross-link in the open conformation (<7A) but are widely separated (>30A) in the closed conformation. Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels. We tested whether drug substrates or inhibitors could inhibit cross-linking of the mutant. It was found that only tariquidar blocked A80C/R741C cross-linking. Tariquidar was also a more potent pharmacological chaperone than other P-gp substrates/modulators such as cyclosporine A. Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Tariquidar interacted with the transmembrane domains because it could rescue a misprocessed truncation mutant lacking the nucleotide-binding domains. These results show that tariquidar is a potent pharmacological chaperone and inhibits P-gp drug efflux by blocking transition to the open state during the catalytic cycle.
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No. Sentence Comment
28 Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp.
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ABCB1 p.Phe804Asp 25456855:28:59
status: NEW68 After 5 h at 37 8C, the medium was replaced with fresh medium with or without 5 mM cyclosporine A or 0.5 mM tariquidar for rescue of mutant F804D and 500 nM tariquidar, 10 mM cyclosporine A, 50 mM verapamil, 20 mM vinblastine or 100 mM capsaicin for rescue of TMD1+2.
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ABCB1 p.Phe804Asp 25456855:68:140
status: NEW185 We previously found that some P-gp mutants such as F804D could not be rescued by any drug substrate [45].
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ABCB1 p.Phe804Asp 25456855:185:51
status: NEW186 The F804D mutation is located in intracellular helix 3 that mediates contact between the third intracellular loop (ICL3) in TMD2 and NBD1.
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ABCB1 p.Phe804Asp 25456855:186:4
status: NEW187 To test if tariquidar could promote maturation of F804D, the A52-tagged mutant was transiently expressed in the presence of 300 nM of the compound for 18 h.
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ABCB1 p.Phe804Asp 25456855:187:50
status: NEW188 Immunoblot analysis of whole cell SDS extracts showed that tariquidar could promote maturation of F804D to yield mature P-gp as about 50% of the steady-state product (Fig. 4C and D).
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ABCB1 p.Phe804Asp 25456855:188:98
status: NEW208 Does tariquidar also inhibit A80C/R741C cross-linking when A B G268V 0 12.5 25 50 100 200 400 800 [Tar] (nM) 170 kDa 150 kDa GAPDH C D 170 kDa 150 kDa GAPDH None Cyclo Tariq F804D 0 20 40 60 80 100 Percent Mature [Tariq] nM 0 12.5 25 50 100 200 400 800 * * * * * * None Cyclo Tariq 0 20 40 60 80 100 Percent Mature * Fig. 4.
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ABCB1 p.Phe804Asp 25456855:208:174
status: NEW215 (C) Whole cell extracts of HEK 293 cells expressing A52-tagged P-gp processing mutant F804D in the absence (None) or presence of 10 mM cyclosporine A (Cyclo) or 300 nM tariquidar (Tariq) were subjected to immunoblot analysis.
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ABCB1 p.Phe804Asp 25456855:215:86
status: NEW306 Perhaps tariquidar acts as a particularly effective pharmacological chaperone to rescue P-gp processing mutants such as F804D (Fig. 4C) because it interacts at both the predicted Rand H-sites to have an additive effect on maturation.
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ABCB1 p.Phe804Asp 25456855:306:120
status: NEW[hide] The Transmission Interfaces Contribute Asymmetrica... J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18. Loo TW, Clarke DM
The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.
J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18., [PMID:25987565]
Abstract [show]
P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.
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No. Sentence Comment
159 For example, tariquidar but not cyclosporine A could repair the F804D mutation at the ICL3-NBD1 interface (41).
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ABCB1 p.Phe804Asp 25987565:159:64
status: NEW