ABCB1 p.Asp805Ala
| Predicted by SNAP2: | A: D (75%), C: D (71%), E: D (59%), F: D (80%), G: D (80%), H: D (80%), I: D (80%), K: D (85%), L: D (80%), M: D (80%), N: D (63%), P: D (85%), Q: D (71%), R: D (80%), S: D (71%), T: D (71%), V: D (80%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: 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, Y: D, |
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[hide] Mutations in intracellular loops 1 and 3 lead to m... J Biol Chem. 2013 Nov 8;288(45):32622-36. doi: 10.1074/jbc.M113.498980. Epub 2013 Sep 24. Kapoor K, Bhatnagar J, Chufan EE, Ambudkar SV
Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70.
J Biol Chem. 2013 Nov 8;288(45):32622-36. doi: 10.1074/jbc.M113.498980. Epub 2013 Sep 24., [PMID:24064216]
Abstract [show]
P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 degrees C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4-6 h) treatment with 15 muM cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.
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No. Sentence Comment
313 We also substituted these two aspartates paired with either Ala (D164A/D805A) or Asn (D164N/D805N) and observed lower cell surface expression in both cases.
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ABCB1 p.Asp805Ala 24064216:313:71
status: NEW314 Single substitution of Asp-164 with Ala (D164A) or Asn (D164N) led to similar expression and function as D164C.3 The substitution of Asp-805 with Ala (D805A) or Asn (D805N) is less drastic as compared with D805C, but the protein still does not localize to the cell surface.3 All these substitutions with either Ala or Asn could be rescued to the cell surface in the presence of CsA (data not shown).
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ABCB1 p.Asp805Ala 24064216:314:133
status: NEWX
ABCB1 p.Asp805Ala 24064216:314:151
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
80 By contrast, the D800A, V801S, S802A, D805A, and D806A mutations in IH3 appeared to have little or no effect on folding as they yielded mature 170-kDa P-gp as their major product.
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ABCB1 p.Asp805Ala 24275649:80:38
status: NEW