ABCB1 p.Asp805Asn
| 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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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.Asp805Asn 24064216:313:92
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.Asp805Asn 24064216:314:166
status: NEW