ABCB1 p.Asp164Cys
| Predicted by SNAP2: | A: D (75%), C: D (75%), E: D (66%), F: D (80%), G: D (80%), H: D (80%), I: D (85%), K: D (85%), L: D (85%), M: D (80%), N: D (75%), P: D (91%), Q: D (75%), R: D (85%), S: D (71%), T: D (75%), V: D (80%), W: D (85%), Y: D (75%), |
| 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
7 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.
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ABCB1 p.Asp164Cys 24064216:7:25
status: NEW29 2 The abbreviations used are: ABC, ATP binding cassette; P-gp, P-glycoprotein; CsA, cyclosporine A; CYH, cycloheximide; ER, endoplasmic reticulum; ICL, intracellular loop; NBD, nucleotide binding domain; NBD-CsA, NBD-cyclosporine A; Rh123, rhodamine 123; SNP, single-nucleotide polymorphism; TMD, transmembrane domain; Endo H, endo-beta-N-acetylgluco- saminidase H; PNGase F, peptide N-glycosidase F; FKBP, FK506-binding protein; DD, D164C/D805C.
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ABCB1 p.Asp164Cys 24064216:29:434
status: NEW35 Our insect cell studies show that the double mutant D164C/D805C displays no change in Km for ATP binding, thus contradicting the suggested direct interaction of these residues with ATP (5).
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ABCB1 p.Asp164Cys 24064216:35:52
status: NEW37 The conserved aspartates, when mutated to cysteine singly (D164C, D805C) or together (D164C/D805C), affected the processing and trafficking of P-gp to the cell membrane.
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ABCB1 p.Asp164Cys 24064216:37:59
status: NEWX
ABCB1 p.Asp164Cys 24064216:37:86
status: NEW38 We discovered that the maturation defect associated with the D164C/D805C mutant was sensitive to growth temperature.
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ABCB1 p.Asp164Cys 24064216:38:61
status: NEW39 When cells expressing the D164C/ D805C mutant were incubated at 27 &#b0;C (similar to growth conditions for High-five insect cells), normal maturation of P-gp was observed.
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ABCB1 p.Asp164Cys 24064216:39:26
status: NEW41 Subsequently, we observed that the incubation of cells expressing the D164C/ D805C mutant in the presence of pharmacological chaperones or substrates such as cyclosporine A (CsA) completely rescued the misfolded protein as a functional protein to the cell surface.
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ABCB1 p.Asp164Cys 24064216:41:70
status: NEW416 Lane 1, cysless-WT; lane 2, D164C/D805C; lane 3, cysless-WT (CsA); lane 4, D164C/D805C (CsA).
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ABCB1 p.Asp164Cys 24064216:416:28
status: NEWX
ABCB1 p.Asp164Cys 24064216:416:75
status: NEW424 Importantly, like èc;F508-CFTR (43), once the D164C/D805C mutant P-gp is rescued and reaches the cell surface, it is functional to the same levels as cysless-WT.
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ABCB1 p.Asp164Cys 24064216:424:50
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
289 To test if residues Asp-164 or Asp-805 were essential for activity, Kapoor et al. (51) tested the effects of introducing D164C or D805C mutations into human Cys-less P-gp.
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ABCB1 p.Asp164Cys 25987565:289:121
status: NEW291 The D164C mutation reduced cell surface expression to about 40% of the Cys-less parent while the presence of both the D164C and D805C mutations reduced cell surface expression to about 20% of the parent.
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ABCB1 p.Asp164Cys 25987565:291:4
status: NEWX
ABCB1 p.Asp164Cys 25987565:291:118
status: NEW292 Immunoblot analysis of whole cell extracts expressing D164C/D805C showed that the major product was immature P-gp.
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ABCB1 p.Asp164Cys 25987565:292:54
status: NEW293 The observation that P-gp mutants R262A/ R905A, T263A/T906A (this study), and D164C/D805C (51) retain substantial activity suggests that there are multiple NBD-TMD contacts as reported by Jin et al. (21).
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ABCB1 p.Asp164Cys 25987565:293:78
status: NEW294 The D164C/D805C mutant could be efficiently rescued when expressed in the presence of cyclosporine A to yield mature P-gp as the major product.
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ABCB1 p.Asp164Cys 25987565:294:4
status: NEW299 The likely explanation is that the D164C mutation was introduced into a Cys-less P-gp background while the D164A mutation was introduced into a wild-type background.
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ABCB1 p.Asp164Cys 25987565:299:35
status: NEW