ABCB1 p.Gly269Val
| Predicted by SNAP2: | A: N (82%), C: N (87%), D: D (71%), E: D (75%), F: D (71%), H: D (59%), I: D (59%), K: D (71%), L: D (63%), M: N (61%), N: N (57%), P: D (75%), Q: N (57%), R: D (63%), S: N (61%), T: N (66%), V: D (63%), W: D (71%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: 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] The human multidrug resistance P-glycoprotein is i... FASEB J. 1999 Oct;13(13):1724-32. Loo TW, Clarke DM
The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.
FASEB J. 1999 Oct;13(13):1724-32., [PMID:10506575]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide cross-linking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.
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No. Sentence Comment
64 Accordingly, the histidine-tagged 150 kDa protein of mutants G268V, G269V, and Y710A were isolated (Fig. 1, lanes 1, 3, and 5).
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ABCB1 p.Gly269Val 10506575:64:68
status: NEW66 To convert the processing mutants G268V, G269V, and Y710A to the mature enzyme, the mutants were synthesized in the presence of 10 M cyclosporin A to induce maturation (19).
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ABCB1 p.Gly269Val 10506575:66:41
status: NEW73 As reported previously (19), the mature enzyme of mutants G268V and G269V had ϳ50 to 75% of the activity of wild-type P-gp.
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ABCB1 p.Gly269Val 10506575:73:68
status: NEW76 ATPase activity of P-gp that is prevented from undergoing maturation It was possible that the 150 kDa core-glycosylated P-gp of the processing mutants G268V, G269V, and Y710A were inactive because the point mutations inherently caused misfolding of the protein and subsequent retention in the endoplasmic reticulum.
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ABCB1 p.Gly269Val 10506575:76:158
status: NEW84 Equivalent amounts of histidine-tagged mature and core-glycosylated P-gp of wild-type and mutants G268V, G269V, and Y710A, isolated by nickel-chelate chromatography, were reconstituted with lipid and assayed for verapamil- (1 mM) and vinblastine- (0.05 mM) stimulated ATPase activities.
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ABCB1 p.Gly269Val 10506575:84:105
status: NEW88 HEK293 cells expressing histidine-tagged P-gp mutants G268V, G269V or Y710A were incubated with (ϩ) or without (-) 10 M cyclosporin A (cyclo) for 24 h. The cells were harvested and solubilized with n-dodecyl-beta-D-maltoside and the cell extracts were subjected to nickel-chelate chromatography with 20 mM imidazole in the wash buffers.
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ABCB1 p.Gly269Val 10506575:88:61
status: NEW[hide] Quality control by proteases in the endoplasmic re... J Biol Chem. 1998 Dec 4;273(49):32373-6. Loo TW, Clarke DM
Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein.
J Biol Chem. 1998 Dec 4;273(49):32373-6., 1998-12-04 [PMID:9829963]
Abstract [show]
Human P-glycoprotein is synthesized in HEK 293 cells as two major products: the 150-kDa core-glycosylated intermediate and the 170-kDa mature proteins. The 150- and 170-kDa proteins were not detected in mutants such as G341C. The major protein in this mutant was a 130-kDa proteolytic degradation product. This result suggested that the mutant protein was misfolded and sensitive to proteolytic digestion during or immediately after synthesis. We found that mutation of Arg113, located in the first extracellular loop of P-glycoprotein and near the consensus glycosylation sites, to Ala, Lys, Glu, Met, or Cys blocked formation of the 130-kDa product. Introduction of R113A into mutant G341C resulted in the synthesis of a mature (170 kDa) and functional transporter. Similarly, when R113A was introduced into misprocessed mutants, there was increased synthesis of the 150-kDa core-glycosylated intermediate. Maturation of the core-glycosylated intermediate into the mature enzyme, however, was not observed. These results suggest that polytopic proteins are accessible to proteases in the lumen of the endoplasmic reticulum during biosynthesis and that proteases are important contributors to the quality control mechanism involved in protein folding. It is also shown that unstable proteins can be made more stable by removal of hypersensitive proteolytic sites.
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117 By contrast, the core-glycosylated G268V protein failed to mature and was almost undetectable by 24 h. In the double mutant (G268V ϩ R113A), however, there was a 5-10-fold increase in the amount of P-gp at 0 h, but the mutant still failed to mature and was degraded by 24 h. A similar pattern was observed for the processing mutant G269V (data not shown).
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ABCB1 p.Gly269Val 9829963:117:338
status: NEW[hide] Human P-glycoprotein contains a greasy ball-and-so... J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.
J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3., [PMID:23733192]
Abstract [show]
The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.
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No. Sentence Comment
96 The I261S, V264S, F267A, G268V, and G269V mutations blocked maturation such that the 150-kDa immature protein was the major product (Fig. 1B).
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ABCB1 p.Gly269Val 23733192:96:36
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
152 Asymmetry of the P-gp Drug Pump Transmission Interfaces JULY 3, 2015ߦVOLUME 290ߦNUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 16957 at SEMMELWEIS UNIV OF MEDICINE on December 4, twenty-eight mutations in ICL2 (A250L, G251V, V253S, A254L, E256A, L258S, I261S, V264S, F267A, G268V, G269V, L274S, R276A, Y277A) inhibited maturation of P-gp (b0d;15% mature P-gp) while four other ICL2 mutations (A2650L, R262A, T263A, and I265S) partially reduced maturation of P-gp (about 55-60% mature P-gp).
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ABCB1 p.Gly269Val 25987565:152:289
status: NEW