ABCB1 p.Trp803Ala
| Predicted by SNAP2: | A: D (85%), C: D (75%), D: D (91%), E: D (91%), F: N (53%), G: D (85%), H: D (91%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (85%), V: D (80%), Y: N (57%), |
| 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, Y: D, |
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[hide] Correction of defective protein kinesis of human P... J Biol Chem. 1997 Jan 10;272(2):709-12. Loo TW, Clarke DM
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem. 1997 Jan 10;272(2):709-12., 1997-01-10 [PMID:8995353]
Abstract [show]
There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.
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No. Sentence Comment
64 In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown).
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ABCB1 p.Trp803Ala 8995353:64:319
status: NEW[hide] Locking intracellular helices 2 and 3 together ina... J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25. Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PMID:24275649]
Abstract [show]
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(beta,gamma-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
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No. Sentence Comment
79 It was Clamping IH2 to IH3 Inhibits P-gp 230 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 1ߦJANUARY 3, found that two mutations in IH3 (W803A and F804A) inhibited maturation of P-gp (Fig. 1, B and C) so that immature 150-kDa P-gp was the major product.
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ABCB1 p.Trp803Ala 24275649:79:155
status: NEW83 Because W803A and F804A mutations in IH3 yielded the immature 150-kDa immature P-gp as the major product, we tested whether the mutants could be rescued with cyclosporine A.
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ABCB1 p.Trp803Ala 24275649:83:8
status: NEW85 Accordingly, the A52-tagged mutants W803A and F804A were expressed in HEK 293 cells in the absence or presence of 5 òe;M cyclosporine A, and whole cell SDS extracts were subjected to immunoblot analysis. Fig. 2A shows that expression of either W803A or F804A in the presence of cyclosporine A yielded the mature 170-kDa P-gp as the major product.
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ABCB1 p.Trp803Ala 24275649:85:36
status: NEWX
ABCB1 p.Trp803Ala 24275649:85:248
status: NEW89 To determine whether the W803A and F804A mutants rescued with cyclosporine A were active, the histidine-tagged mutants W803A and F804A were expressed in HEK 293 cells in the presence of cyclosporine A, and P-gp was isolated by nickel-chelate chromatography.
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ABCB1 p.Trp803Ala 24275649:89:25
status: NEWX
ABCB1 p.Trp803Ala 24275649:89:119
status: NEW101 Drug rescue of processing mutants and activity of IH3 mutants W803A and F804A.
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ABCB1 p.Trp803Ala 24275649:101:62
status: NEW102 A52-tagged WT P-gp, mutants W803A and F804A (A), or IH3 flanking mutants (F793A, L797A, L814A, and L818A) (B) were expressed in HEK293cellsintheabsence(afa;)orpresence(af9;)of5òe;M cyclosporineA(Cyclo) for 18 h, and whole cell extracts were subjected to immunoblot analysis.
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ABCB1 p.Trp803Ala 24275649:102:28
status: NEW103 The positions of mature (170-kDa) and immature (150-kDa) forms of P-gp are shown. C, verapamil-stimulated ATPase activities of histidine-tagged wild-type P-gp and W803A and F804A mutants.
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ABCB1 p.Trp803Ala 24275649:103:163
status: NEW