ABCB1 p.Arg741Cys
| Predicted by SNAP2: | A: N (82%), C: N (66%), D: N (66%), E: N (72%), F: N (61%), G: N (78%), H: N (78%), I: N (78%), K: N (87%), L: N (82%), M: N (66%), N: N (82%), P: N (78%), Q: N (78%), S: N (93%), T: N (87%), V: N (72%), W: D (53%), Y: N (82%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Cysteines introduced into extracellular loops 1 an... J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22. Loo TW, Clarke DM
Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22., [PMID:25053414]
Abstract [show]
P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 A apart) because they are separated widely (30.4 A apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.
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No. Sentence Comment
51 Mutations A80C, R741C, or A80C plus R741C were introduced into A52-tagged Cys-less P-gp or A52- or 10-histidine-tagged wild-type P-gps as described previously (25).
X
ABCB1 p.Arg741Cys 25053414:51:16
status: NEWX
ABCB1 p.Arg741Cys 25053414:51:36
status: NEW65 Drug Resistance Assays-Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutant A80C/R741C were generated as described previously (33).
X
ABCB1 p.Arg741Cys 25053414:65:107
status: NEW73 Cross-linking of Mutant A80C/R741C in the Presence or Absence of ATP-HEK 293 cells were transiently transfected with the A52-tagged mutant A80C/R741C (in a Cys-less background) and then grown for 48 h at 30 &#b0;C to promote maturation of P-gp.
X
ABCB1 p.Arg741Cys 25053414:73:29
status: NEWX
ABCB1 p.Arg741Cys 25053414:73:144
status: NEW82 P-gp Is Inhibited by Extracellular Loop Cross-linking 24750 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER RESULTS Cysteines Introduced into ECL 1 (A80C) and ECL 4 (R741C) of Cys-less P-gp Cross-link Spontaneously-The major goal of the study was to use disulfide cross-linking to test whether we could detect the open conformation of P-gp in intact cells and whether locking P-gp in an open conformation would have an effect on its activity.
X
ABCB1 p.Arg741Cys 25053414:82:175
status: NEW89 To test whether cysteines introduced at positions 80 and 741 would cross-link, mutants A80C, R741C, and A80C/R741C were constructed in a Cys-less P-gp background.
X
ABCB1 p.Arg741Cys 25053414:89:93
status: NEWX
ABCB1 p.Arg741Cys 25053414:89:109
status: NEW97 Mutants A80C, R741C, and A80C/R741C resembled the Cys-less parent because the 150-kDa immature protein was the major product when expressed in the absence of cyclosporine A (Fig. 2A).
X
ABCB1 p.Arg741Cys 25053414:97:14
status: NEWX
ABCB1 p.Arg741Cys 25053414:97:30
status: NEW98 Cyclosporine A promoted maturation of the Cys-less, A80C, and R741C mutants to yield mature 170-kDa P-gp as the major product (Fig. 2A).
X
ABCB1 p.Arg741Cys 25053414:98:62
status: NEW99 By contrast, mutant A80C/R741C in the presence of cyclosporine A yielded a protein that migrated with slower mobility than the mature 170-kDa P-gp (Fig. 2A).
X
ABCB1 p.Arg741Cys 25053414:99:25
status: NEW112 Cysteines A80C/R741C in a Cys-less background cross-link when the mutant was rescued with a drug substrate.
X
ABCB1 p.Arg741Cys 25053414:112:15
status: NEW113 A, A52-tagged Cys-less P-gp (C-less) and mutants A80C, R741C, or A80C/R741C were transiently expressed in HEK 293 cells in the absence (afa;) or presence (af9;) of 5 òe;M cyclosporine A (Cyclo).
X
ABCB1 p.Arg741Cys 25053414:113:55
status: NEWX
ABCB1 p.Arg741Cys 25053414:113:70
status: NEW114 Whole cell SDS extracts (containing no thiol-reducing agent) were subjected to immunoblot analysis. B, whole cells SDS extracts of mutant A80C/R741C grown in the presence of cyclosporine A were treated without (afa;) or with (af9;) 10 mM DTT prior to immunoblot analysis.
X
ABCB1 p.Arg741Cys 25053414:114:143
status: NEW115 The posi- tionsofcross-linked(X-link),mature(170-kDa),andimmature(150-kDa)P-gps are indicated. P-gp Is Inhibited by Extracellular Loop Cross-linking SEPTEMBER 5, 2014ߦVOLUME 289ߦNUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 24751 at SEMMELWEIS UNIV OF MEDICINE on December 12, was supported by the observation that treatment of mutant A80C/R741C grown in cyclosporine A with 10 mM DTT converted the slow migrating P-gp protein into the 170-kDa protein (Fig. 2B).
X
ABCB1 p.Arg741Cys 25053414:115:350
status: NEW116 The results suggest that mature 170-kDa A80C/ R741C protein efficiently cross-links when expressed in the presence of cyclosporine A.
X
ABCB1 p.Arg741Cys 25053414:116:46
status: NEW117 Cysteines A80C (ECL1) and R741C (ECL4) Can Cross-link Spontaneously in the Absence of Drug Substrates-Because maturation of mutant A80C/R741C in the Cys-less background required the presence of cyclosporine A, we wanted to determine whether this mutant, when introduced into a wild-type background, would cross-link spontaneously in the absence of cyclosporine A.
X
ABCB1 p.Arg741Cys 25053414:117:26
status: NEWX
ABCB1 p.Arg741Cys 25053414:117:136
status: NEW119 Accordingly, mutations A80C, R741C, and A80C/R741C were introduced into A52-tagged wild-type P-gp.
X
ABCB1 p.Arg741Cys 25053414:119:29
status: NEWX
ABCB1 p.Arg741Cys 25053414:119:45
status: NEW122 The expression of mutants A80C and R741C also resembled wild-type P-gp in that the major product was the 170-kDa protein.
X
ABCB1 p.Arg741Cys 25053414:122:35
status: NEW123 The major product in mutant A80C/R741C, however, was a protein that migrated with slower mobility than the mature 170-kDa slower-migrating protein (Fig. 3).
X
ABCB1 p.Arg741Cys 25053414:123:33
status: NEW124 Treatment of the slowly migrating A80C/ R741C protein with 10 mM DTT yielded the 170-kDa protein (Fig. 3).
X
ABCB1 p.Arg741Cys 25053414:124:40
status: NEW126 It is possible that mutant A80C/R741C showed efficient cross-linking because it was transiently expressed in HEK 293 cells.
X
ABCB1 p.Arg741Cys 25053414:126:32
status: NEW127 We then tested whether mutant A80C/R741C was also efficiently cross-linked if it was stably expressed in BHK cells.
X
ABCB1 p.Arg741Cys 25053414:127:35
status: NEW128 Accordingly, BHK cells were cotransfected with A52-tagged wild-type P-gp or mutant A80C/R741C (in a wild-type background) cDNAs and a plasmid containing a neomycin marker (pWL-neo).
X
ABCB1 p.Arg741Cys 25053414:128:88
status: NEW133 By contrast, the major products in mutant A80C/R741C were the slowly migrating cross-linked and the 150-kDa proteins.
X
ABCB1 p.Arg741Cys 25053414:133:47
status: NEW134 It appeared that the mature 170-kDa A80C/R741C protein was cross-linked efficiently because little (b0d;5% of total P-gp) of the mature 170-kDa protein was present (Fig. 4, right panel).
X
ABCB1 p.Arg741Cys 25053414:134:41
status: NEW136 Whole cell SDS extracts from cells expressing wild-type P-gp or mutant A80C/ R741C were treated with endoglycosidases that cleaved only core sugars (endoglycosidase H) or all carbohydrate (PNGase F).
X
ABCB1 p.Arg741Cys 25053414:136:77
status: NEW138 By contrast, the 150-kDa A80C/R741C immature protein, but not the cross-linked product, was sensitive to endoglycosidase H.
X
ABCB1 p.Arg741Cys 25053414:138:30
status: NEW139 The cross-linked A80C/R741C protein was sensitive only to PNGase F (Fig. 4, right panel).
X
ABCB1 p.Arg741Cys 25053414:139:22
status: NEW142 Cysteines A80C (ECL1) and R741C (ECL4) Can Cross-link Spontaneously After Synthesis and Maturation of P-gp-Disulfide bonds are formed in the endoplasmic reticulum of eukaryotic cells (reviewed in Ref. 39).
X
ABCB1 p.Arg741Cys 25053414:142:26
status: NEW143 It is possible that A80C/R741C may form a disulfide bond during synthesis in the endoplasmic reticulum but that the cross-linked protein did not run with altered mobility in SDS-PAGE gels so that it was not detected.
X
ABCB1 p.Arg741Cys 25053414:143:25
status: NEW144 A way to overcome this problem was to test whether the disulfide bond in mutant A80C/R741C could reform if synthesis of new P-gp was blocked and cross-linked P-gp at the cell surface was reduced.
X
ABCB1 p.Arg741Cys 25053414:144:85
status: NEW145 Accordingly, BHK cells expressing A52-tagged A80C/ R741C P-gp were pretreated for 1 h with cycloheximide to inhibit protein synthesis (40).
X
ABCB1 p.Arg741Cys 25053414:145:51
status: NEW146 The cells were then treated briefly (1 min) with 5 mM DTT to reduce the A80C/R741C disulfide bond at the cell surface.
X
ABCB1 p.Arg741Cys 25053414:146:77
status: NEW152 Cysteines A80C/R741C in a wild-type background cross-link in the absence of drug substrates.
X
ABCB1 p.Arg741Cys 25053414:152:15
status: NEW153 A52-tagged WT P-gp and mutants A80C, R741C, or A80C/R741C were transiently expressed in HEK 293 cells in the absence of drug substrates.
X
ABCB1 p.Arg741Cys 25053414:153:37
status: NEWX
ABCB1 p.Arg741Cys 25053414:153:52
status: NEW157 Endoglycosidase digestion of wild-type and mutant A80C/ R741C P-gp expressed in BHK cells.
X
ABCB1 p.Arg741Cys 25053414:157:56
status: NEW158 Whole cell SDS extracts of BHK cells expressing A52-tagged wild-type P-gp or mutant A80C/R741C (wild-type background) that were grown without drug substrates were treated without (afa;)orwith(af9;)endoglycosidaseHf (H)orPNGaseF(F)andsubjectedtoimmu- noblot analysis.
X
ABCB1 p.Arg741Cys 25053414:158:89
status: NEW159 The positions of cross-linked (X-link), mature (170-kDa), immature (150-kDa), and unglycosylated (140-kDa) P-gps are indicated. P-gp Is Inhibited by Extracellular Loop Cross-linking 24752 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER About 50% of mutant A80C/R741C was cross-linked by 18 min and, at 54 min, the amount of cross-linked P-gp resembled that of the untreated control (Fig. 5).
X
ABCB1 p.Arg741Cys 25053414:159:270
status: NEW161 The A80C/R741C Disulfide Bond Reduces P-gp Drug-stimulated ATPase Activity-To test whether the A80C/R741C mutant was active, histidine-tagged wild-type P-gp or mutant A80C/R741C were transiently expressed in HEK 293 cells.
X
ABCB1 p.Arg741Cys 25053414:161:9
status: NEWX
ABCB1 p.Arg741Cys 25053414:161:100
status: NEWX
ABCB1 p.Arg741Cys 25053414:161:172
status: NEW165 Cross-linking of A80C and R741C reduced activity severely.
X
ABCB1 p.Arg741Cys 25053414:165:26
status: NEW167 It was found however, that the verapamil-stimulated ATPase activity of mutant A80C/R741C was not significantly different from wild-type P-gp when assayed in the presence of DTT (Fig. 6).
X
ABCB1 p.Arg741Cys 25053414:167:83
status: NEW169 The A80C/R741C Disulfide Bond Inhibits Drug Efflux-We then tested whether mutant A80C/R741C could confer drug resistance.
X
ABCB1 p.Arg741Cys 25053414:169:9
status: NEWX
ABCB1 p.Arg741Cys 25053414:169:86
status: NEW172 We first tested whether the A80C/R741C disulfide bond would affect the ability of P-gp to confer drug resistance on BHK cells.
X
ABCB1 p.Arg741Cys 25053414:172:33
status: NEW173 BHK control cells and cells expressing equivalent levels of wild-type or mutant A80C/R741C P-gp were incubated with various concentrations of vinblastine or colchicine to determine the concentration needed to inhibit cell growth.
X
ABCB1 p.Arg741Cys 25053414:173:85
status: NEW176 By contrast, for cells expressing mutant A80C/R741C, the LD50s occurred at 2.3 and 0.6 nM (3-fold-increase in resistance) and 1.8 and 0.2 òe;M (about 3-fold increase in resistance) for vinblastine and colchicine, respectively.
X
ABCB1 p.Arg741Cys 25053414:176:46
status: NEW178 These results suggest that the presence of the A80C/R741C disulfide bond inhibited drug efflux.
X
ABCB1 p.Arg741Cys 25053414:178:52
status: NEW179 Reduction of the A80C/R741C Disulfide Bond Promotes Drug Efflux-Would reduction of the A80C/R741C disulfide bond increase the ability of P-gp to confer drug resistance?
X
ABCB1 p.Arg741Cys 25053414:179:22
status: NEWX
ABCB1 p.Arg741Cys 25053414:179:92
status: NEW182 We also found that significant reduction of the A80C/R741C bond required 100-fold (30 mM) higher levels of beta-mercaptoethanol.3 An alternative approach to reduce the A80C/R741C disulfide bond was be to use DTT.
X
ABCB1 p.Arg741Cys 25053414:182:53
status: NEWX
ABCB1 p.Arg741Cys 25053414:182:173
status: NEW184 Accordingly, BHK cells expressing A52-tagged mutant A80C/R741C were incubated with medium containing various concentrations of DTT.
X
ABCB1 p.Arg741Cys 25053414:184:57
status: NEW189 We then tested whether mutant A80C/R741C was able to confer resistance to vinblastine when incubated in the presence 3 T. W. Loo and D. M. Clarke, unpublished observations.
X
ABCB1 p.Arg741Cys 25053414:189:35
status: NEW191 Mature A80C/R741C P-gp cross-links after treatment with DTT.
X
ABCB1 p.Arg741Cys 25053414:191:12
status: NEW192 BHK cells expressing A52-tagged mutant A80C/R741C (wild-type background) were incubated for 1 h in the presence of 0.5 mg/ml cycloheximide.
X
ABCB1 p.Arg741Cys 25053414:192:44
status: NEW198 Cross-linking of mutant A80C/R741C inhibits verapamil-stimulated ATPase activity.
X
ABCB1 p.Arg741Cys 25053414:198:29
status: NEW199 Histidine-tagged P-gp (WT) or mutant A80C/R741C (in a wild-type background) was transiently expressed in HEK 293 cells.
X
ABCB1 p.Arg741Cys 25053414:199:42
status: NEW203 Accordingly, control BHK cells and BHK cells expressing equivalent levels of wild-type or mutant A80C/R741C P-gp were incubated in the presence of various concentrations of vinblastine and 1.1 mM DTT.
X
ABCB1 p.Arg741Cys 25053414:203:102
status: NEW204 Because this assay required about 6 days, it was necessary to change the medium daily because the half-life of DTT is only several hours (47) and because the reduced A80C/R741C mutant could readily reform the cross-link (Fig. 5).
X
ABCB1 p.Arg741Cys 25053414:204:171
status: NEW205 It was found that wild-type P-gp conferred a similar level of resistance to vinblastine in the presence of DTT (about 60-fold) (Fig. 8B), whereas mutant A80C/R741C showed an approximately 25-fold increase in the ability to confer resistance (10-fold increase compared with the absence of DTT).
X
ABCB1 p.Arg741Cys 25053414:205:158
status: NEW206 The results indicate that mutant A80C/R741C can confer relatively high levels of drug resistance when the disulfide bond is reduced.
X
ABCB1 p.Arg741Cys 25053414:206:38
status: NEW207 Cysteines A80C/R741C Cross-link rapidly in the Absence or Presence of ATP-Spontaneous cross-linking of mutant A80C/ R741C after treatment of the cells with 5 mM DTT was relatively slow because about 50% cross-linking occurred after 18 min at 37 &#b0;C (Fig. 5).
X
ABCB1 p.Arg741Cys 25053414:207:15
status: NEWX
ABCB1 p.Arg741Cys 25053414:207:116
status: NEW210 We first tested whether ATP hydrolysis was required for spontaneous cross-linking of A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:210:90
status: NEW213 HEK 293 cells were then transfected with A52-tagged wild-type P-gp, mutant A80C/R741C (in a wild-type background), or A80C/R741C/E556Q/E1201Q (in a wild-type background).
X
ABCB1 p.Arg741Cys 25053414:213:80
status: NEWX
ABCB1 p.Arg741Cys 25053414:213:123
status: NEW216 Both the A80C/R741C and A80C/R741C/E556Q/E1201Q mutants yielded cross-linked P-gp as the major product.
X
ABCB1 p.Arg741Cys 25053414:216:14
status: NEWX
ABCB1 p.Arg741Cys 25053414:216:29
status: NEW218 These results indicate that ATP hydrolysis was not essential for cross-linking mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:218:91
status: NEW219 We then determined whether spontaneous cross-linking of A80C/R741C and A80C/R741C/E556Q/E1201Q mutants was inefficient by testing the effect of an oxidant (copper phenanthroline) to catalyze formation of the disulfide bond at a reduced temperature.
X
ABCB1 p.Arg741Cys 25053414:219:61
status: NEWX
ABCB1 p.Arg741Cys 25053414:219:76
status: NEW220 HEK 293 cells were transfected with the A52-tagged mutants A80C/R741C and A80C/R741C/E556Q/ E1201Q (in a wild-type background).
X
ABCB1 p.Arg741Cys 25053414:220:64
status: NEWX
ABCB1 p.Arg741Cys 25053414:220:79
status: NEW224 Cross-linking of A80C/R741C inhibits P-gp-mediated drug resistance.
X
ABCB1 p.Arg741Cys 25053414:224:22
status: NEW225 BHK cells expressing no P-gp, A52-tagged wild-type P-gp (WT) or mutant A80C/R741C (in a wild-type background) were incubated in the presence of various concentrations of vinblastine or colchicine to determine the LD50 concentration.
X
ABCB1 p.Arg741Cys 25053414:225:76
status: NEW230 Expression of cells with DTT enhances the ability of mutant A80C/R741C to confer drug resistance.
X
ABCB1 p.Arg741Cys 25053414:230:65
status: NEW231 A, BHK cells expressing A52-tagged mutant A80C/R741C (in a wild-type background) were incubated for 18 h in the presence of various concentrations of DTT.
X
ABCB1 p.Arg741Cys 25053414:231:47
status: NEW234 B, BHK cells expressing A52-tagged wild-type P-gp (WT), mutant A80C/R741C, or no P-gp were incubated in the presence of various concentrations of vinblastine and 1.1 mM DTT to determine the LD50 concentration.
X
ABCB1 p.Arg741Cys 25053414:234:68
status: NEW238 P-gp Is Inhibited by Extracellular Loop Cross-linking 24754 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER stopped by addition of SDS sample buffer containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis. Fig. 9B shows that treatment of DTT-treated cells with oxidant almost completely cross-linked the mature 170-kDa protein in both mutants, A80C/R741C and A80C/R741C/E556Q/E1201Q, within 1 min.
X
ABCB1 p.Arg741Cys 25053414:238:398
status: NEWX
ABCB1 p.Arg741Cys 25053414:238:413
status: NEW240 Next, we tested whether ATP binding was required for cross-linking of mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:240:82
status: NEW241 This required the use of membranes prepared from cells expressing mutant A80C/R741C in the Cys-less background.
X
ABCB1 p.Arg741Cys 25053414:241:78
status: NEW243 Accordingly, HEK 293 cells were transfected with the A52-tagged mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:243:76
status: NEW251 Therefore, we tested whether vanadate trapping of nucleotides could inhibit cross-linking of mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:251:105
status: NEW252 Membranes were prepared from HEK 293 cells expressing A52-tagged A80C/R741C that were treated with DTT.
X
ABCB1 p.Arg741Cys 25053414:252:70
status: NEW254 The reactions were stopped by addition of 2afb; SDS sample buffer containing no thiol-reducing agent. Samples were then subjected to immunoblot analysis. Fig. 10B shows that vanadate trapping of nucleotides inhibits cross-linking of mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:254:248
status: NEW255 DISCUSSION We found that linking the P-gp halves by direct cross-linking of extracellular cysteines A80C/R741C inhibited ATPase activity.
X
ABCB1 p.Arg741Cys 25053414:255:105
status: NEW260 Cross-linking of mutant A80C/R741C does not require ATP hydrolysis and is rapid.
X
ABCB1 p.Arg741Cys 25053414:260:29
status: NEW261 A, HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants A80C/R741C (in a wild-type background), or A80C/ R741C/E556Q/E1201Q.
X
ABCB1 p.Arg741Cys 25053414:261:79
status: NEWX
ABCB1 p.Arg741Cys 25053414:261:123
status: NEW263 Samples were then subjected to immunoblot analysis. B, HEK 293 cells expressing A52-tagged mutants A80C/R741C or A80C/R741C/E556Q/E1201Q (in a wild-type background) were treated with 10 mM DTT for 3 min.
X
ABCB1 p.Arg741Cys 25053414:263:104
status: NEWX
ABCB1 p.Arg741Cys 25053414:263:118
status: NEW268 ATP is not required for cross-linking mutant A80C/R741C.
X
ABCB1 p.Arg741Cys 25053414:268:50
status: NEW269 A, HEK 293 cells expressing A52-tagged mutant A80C/R741C (in a Cys-less background) were treated with 10 mM DTT for 3 min.
X
ABCB1 p.Arg741Cys 25053414:269:51
status: NEW280 Direct linkage of A80C and R741C would favor the open conformation (Fig. 1B).
X
ABCB1 p.Arg741Cys 25053414:280:27
status: NEW285 In agreement with the mouse P-gp studies, we found that ATP did not cause any detectable changes in A80C/R741C cross-linking efficiency (Fig. 10).
X
ABCB1 p.Arg741Cys 25053414:285:105
status: NEW303 Inhibition of P-gp by cross-linking of A80C/R741C was different from antibody inhibition because both ATPase activity and drug efflux were inhibited.
X
ABCB1 p.Arg741Cys 25053414:303:44
status: NEW[hide] Tariquidar inhibits P-glycoprotein drug efflux but... Biochem Pharmacol. 2014 Dec 15;92(4):558-66. doi: 10.1016/j.bcp.2014.10.006. Epub 2014 Oct 22. Loo TW, Clarke DM
Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation.
Biochem Pharmacol. 2014 Dec 15;92(4):558-66. doi: 10.1016/j.bcp.2014.10.006. Epub 2014 Oct 22., [PMID:25456855]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is a drug pump that confers multidrug resistance. Inhibition of P-gp would improve chemotherapy. Tariquidar is a potent P-gp inhibitor but its mechanism is unknown. Here, we tested our prediction that tariquidar inhibits P-gp cycling between the open and closed states during the catalytic cycle. Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Residues A80C/R741C come close enough (<7A) to spontaneously cross-link in the open conformation (<7A) but are widely separated (>30A) in the closed conformation. Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels. We tested whether drug substrates or inhibitors could inhibit cross-linking of the mutant. It was found that only tariquidar blocked A80C/R741C cross-linking. Tariquidar was also a more potent pharmacological chaperone than other P-gp substrates/modulators such as cyclosporine A. Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Tariquidar interacted with the transmembrane domains because it could rescue a misprocessed truncation mutant lacking the nucleotide-binding domains. These results show that tariquidar is a potent pharmacological chaperone and inhibits P-gp drug efflux by blocking transition to the open state during the catalytic cycle.
Comments [show]
None has been submitted yet.
No. Sentence Comment
22 Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C).
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ABCB1 p.Arg741Cys 25456855:22:146
status: NEW23 Residues A80C/R741C come close enough (<7 A da; ) to spontaneously cross-link in the open conformation (<7 A da; ) but are widely separated (>30 A da; ) in the closed conformation.
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ABCB1 p.Arg741Cys 25456855:23:14
status: NEW24 Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels.
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ABCB1 p.Arg741Cys 25456855:24:22
status: NEW26 It was found that only tariquidar blocked A80C/ R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:26:48
status: NEW44 P-gp can also be trapped in an open conformation by cross-linking cysteines introduced into extracellular loops (ECL)1 (A80C) and 4 (R741C).
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ABCB1 p.Arg741Cys 25456855:44:133
status: NEW45 Residues A80C/R741C come close enough to spontaneously cross-link in the open conformation (6.9 A da; , all distances represent predicted a a carbon distances) but are widely separated in the closed conformation (>30 A da; ).
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ABCB1 p.Arg741Cys 25456855:45:14
status: NEW46 The presence of an A80C/R741C disulfide bond inhibits ATPase activity and drug efflux [29].
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ABCB1 p.Arg741Cys 25456855:46:24
status: NEW48 In this study, we determined whether tariquidar blocks formation of the open conformation of P-gp by testing if it inhibits A80C/R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:48:129
status: NEW74 Purification of P-gp and assay of ATPase activity Histidine-tagged A80C/R741C P-gp in the wild-type background was expressed in HEK 293 cells and then isolated by nickel-chelate chromatography as described previously [37].
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ABCB1 p.Arg741Cys 25456855:74:72
status: NEW80 Effect of substrates and inhibitors on A80C/R741C cross-linking BHK cells stably expressing A52-tagged mutant A80C/R741C in the wild-type background [29] were pre-treated for 5 min at 20 8C with PBS containing 10 mM dithiothreitol (to reduce the disulfide bond) in the presence of the following substrate or inhibitor: 500 nM tariquidar, 5 mM cyclosporine A, 10 mM vinblastine, 25 mM verapamil, 25 mM Taxol, 25 mM rhodamine B, 25 mM Hoechst 33342, 25 mM ketoconazole, 10 mM reserpine, 10 mM cis-flupentixol, 10 mM trans-flupentixol or no drug substrate/ inhibitor. Cells were then washed four times with PBS without dithiothreitol but containing the same substrate or inhibitor and then treated with or without 0.1 mM copper phenanthroline in the presence of the same substrate or inhibitor for 3 min at 20 8C.
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ABCB1 p.Arg741Cys 25456855:80:44
status: NEWX
ABCB1 p.Arg741Cys 25456855:80:115
status: NEW92 Tariquidar inhibits spontaneous cross-linking of A80C/R741C P-gp in intact cells We previously showed that cysteines A80C/R741C spontaneously cross-linked when the mutant was expressed in cells [29].
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ABCB1 p.Arg741Cys 25456855:92:54
status: NEWX
ABCB1 p.Arg741Cys 25456855:92:122
status: NEW94 Cysteine A80C is located in ECL1 in the N-half of the protein whereas cysteine R741C is located in ECL4 in the C-half (Fig. 1).
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ABCB1 p.Arg741Cys 25456855:94:79
status: NEW98 Therefore, A80C/R741C cross-linking would be expected when P-gp adopts an open conformation during its catalytic cycle since a disulfide bond spans a distance of about 6-7 A da; .
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ABCB1 p.Arg741Cys 25456855:98:16
status: NEW99 The structure of tariquidar is shown in Fig. 2A. Our goal was to test if tariquidar would affect A80C/R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:99:102
status: NEW100 The first step was to determine if tariquidar could still interact with P-gp after introduction of the A80C and R741C mutations.
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ABCB1 p.Arg741Cys 25456855:100:112
status: NEW102 To test if tariquidar could activate the ATPase activity of A80C/R741C P-gp, the histidine-tagged mutant (in the wild-type background) was transiently expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Arg741Cys 25456855:102:65
status: NEW105 Next, we tested the effect of tariquidar on A80C/R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:105:49
status: NEW106 The A52-tagged A80C/R741C mutant (in the wild-type background) was transiently expressed in HEK 293 cells for 18 h in the absence or presence of various concentrations of tariquidar.
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ABCB1 p.Arg741Cys 25456855:106:20
status: NEW111 Locations of the A80C and R741C mutations and composition of truncation mutants of P-gp.
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ABCB1 p.Arg741Cys 25456855:111:26
status: NEW117 Inhibition of spontaneous A80C/R741C cross-linking in whole cells by tariquidar.
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ABCB1 p.Arg741Cys 25456855:117:31
status: NEW119 (B) Isolated histidine-tagged P-gp mutant A80C/R741C (in wild-type background) was mixed with lipid and ATPase activity was determined in the absence or presence of 1 mM tariquidar.
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ABCB1 p.Arg741Cys 25456855:119:47
status: NEW122 (C) Whole SDS (no reducing agent) cell extracts of HEK 293 cells expressing mutant A52-tagged A80C/R741C (wild-type background) in the presence of various concentrations of tariquidar (Tariq) were subjected to immunoblot analysis.
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ABCB1 p.Arg741Cys 25456855:122:99
status: NEW134 The results show that tariquidar is a potent inhibitor of A80C/ R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:134:64
status: NEW135 By contrast, we previously observed that P-gp substrates such as vinblastine or cyclosporine A did not inhibit spontaneous cross-linking of the A80C/R741C mutant [29].
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ABCB1 p.Arg741Cys 25456855:135:149
status: NEW137 Tariquidar inhibits rapid A80C/R741C cross-linking in the presence of oxidant In a previous study [29], we found that spontaneous cross-linking of mutant A80C/R741C was slow relative to cycling of P-gp through its reaction cycle.
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ABCB1 p.Arg741Cys 25456855:137:31
status: NEWX
ABCB1 p.Arg741Cys 25456855:137:159
status: NEW138 When cells expressing the A80C/R741C mutant were treated with dithiothreitol to reduce the extracellular disulfide bond, it took about 20 min to achieve 50% cross-linking of the mutant after dithiothreitol was removed.
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ABCB1 p.Arg741Cys 25456855:138:31
status: NEW141 Therefore, it was also important to test if tariquidar would also inhibit rapid cross-linking of mutant A80C/R741C in the presence of oxidant.
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ABCB1 p.Arg741Cys 25456855:141:109
status: NEW142 We tested if tariquidar would inhibit A80C/R741C cross-linking in the presence of oxidant using BHK cells stably expressing the mutant [29].
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ABCB1 p.Arg741Cys 25456855:142:43
status: NEW143 Stably transfected BHK cells expressing A80C/R741C P-gp (in the wild-type background) were used rather than transiently transfected HEK 293 cells because they remain attached to the plates during the multiple washing steps required for the oxidant cross-linking assays.
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ABCB1 p.Arg741Cys 25456855:143:45
status: NEW144 We first treated the cells expressing P-gp A80C/R741C with 10 mM dithiothreitol to reduce the disulfide bond located on the extracellular surface.
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ABCB1 p.Arg741Cys 25456855:144:48
status: NEW149 Cross-linked A80C/R741C was the major product (about 80% of total P-gp protein) detected when untreated whole cell extracts of BHK cells expressing the mutant were subjected to immunoblot analysis (Fig. 3A and B, lane A).
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ABCB1 p.Arg741Cys 25456855:149:18
status: NEW153 The results show that tariquidar blocked both slow (Fig. 2C) and fast (Fig. 3A and B, lane D) cross-linking of mutant A80C/R741C.
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ABCB1 p.Arg741Cys 25456855:153:123
status: NEW154 To test the effects of other drug substrates or modulators on cross-linking of mutant A80C/R741C in the presence of oxidant, the cells were treated for 5 min with 10 mM dithiothreitol in the presence of drug substrates such as vinblastine, verapamil, paclitaxel, rhodamine B, or Hoechst 33342 or inhibitors/ modulators such as cyclosporine A, ketoconazole, reserpine, or the cisor trans-isomers of flupentixol.
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ABCB1 p.Arg741Cys 25456855:154:91
status: NEW157 The results show that inhibition of rapid A80C/R741C cross-linking was specific for tariquidar.
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ABCB1 p.Arg741Cys 25456855:157:47
status: NEW161 Tariquidar acts as a noncompetitive inhibitor for drug binding and differed from drug substrates because it was the only compound that blocked A80C/R741C cross-linking (Fig. 3).
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ABCB1 p.Arg741Cys 25456855:161:148
status: NEW166 Only tariquidar inhibited A80C/R741C cross-linking in the presence of oxidant.
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ABCB1 p.Arg741Cys 25456855:166:31
status: NEW167 (A) BHK cells stably expressing A52-tagged mutant A80C/R741C (in wild-type background) were treated without ( ) or with (+) 10 mM dithiothreitol (DTT) to reduce the disulfide bond.
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ABCB1 p.Arg741Cys 25456855:167:55
status: NEW208 Does tariquidar also inhibit A80C/R741C cross-linking when A B G268V 0 12.5 25 50 100 200 400 800 [Tar] (nM) 170 kDa 150 kDa GAPDH C D 170 kDa 150 kDa GAPDH None Cyclo Tariq F804D 0 20 40 60 80 100 Percent Mature [Tariq] nM 0 12.5 25 50 100 200 400 800 * * * * * * None Cyclo Tariq 0 20 40 60 80 100 Percent Mature * Fig. 4.
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ABCB1 p.Arg741Cys 25456855:208:34
status: NEW223 We previously found that complex carbohydrate was required to cause the cross-linked A80C/R741C full-length protein to migrate slower on SDS-PAGE gels [29].
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ABCB1 p.Arg741Cys 25456855:223:90
status: NEW224 Removal of the carbohydrate from the cross-linked A80C/R741C mutant with endoglycosidase F yielded a product that migrated in the same position as wild-type P-gp lacking carbohydrate.
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ABCB1 p.Arg741Cys 25456855:224:55
status: NEW232 The A52-tagged A80C/N-half and R741C/C-half cDNAs (in the Cys-less background) were constructed.
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ABCB1 p.Arg741Cys 25456855:232:31
status: NEW233 The A80C/N-half and R741C/C-half proteins were co-expressed in the absence or presence of 10 mM cyclosporine A or 500 nM tariquidar.
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ABCB1 p.Arg741Cys 25456855:233:20
status: NEW237 To test for A80C/R741C cross-linking we expressed A52-tagged R741C/C-half P-gp with untagged A80C/N-half P-gp in the absence or presence of 10 mM cyclosporine A or 500 nM tariquidar.
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ABCB1 p.Arg741Cys 25456855:237:17
status: NEWX
ABCB1 p.Arg741Cys 25456855:237:61
status: NEW238 The presence of an epitope tag on just the C-half protein was used to make it simpler to detect cross-linking between the R741C/C-half protein and A80C/N-half protein.
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ABCB1 p.Arg741Cys 25456855:238:122
status: NEW259 (A) Whole cell SDS extracts of HEK 293 cells (containing 10 mM dithiothreitol) expressing A52-tagged A80C/N-half and A52-tagged R741C/C-half Pgp (in Cys-less background) in the absence (None) or presence of 10 mM cyclosporine A (Cyclo) or 500 nM tariquidar (Tariq) were subjected to immunoblot analysis.
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ABCB1 p.Arg741Cys 25456855:259:128
status: NEW260 The positions of R741C/C-half or mature or immature forms of A80C/N-half P-gp`s are indicated.
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ABCB1 p.Arg741Cys 25456855:260:17
status: NEW264 (C) Whole cell extracts of cells expressing A52-tagged R741C/C-half P-gp together with untagged A80C/N-half P-gp (in Cys-less background) in the absence (None) or presence of 10 m; cyclosporine A (Cyclo) or 500 nM tariquidar (Tariq) before ( ) or after (+) treatment with 10 mM dithiothreitol (DTT) were subjected to immunoblot analysis with monoclonal antibody A52.
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ABCB1 p.Arg741Cys 25456855:264:55
status: NEW265 The positions of R741C/C-half cross-linked to mature (Mat) and immature (Immat) forms of A80C/N-half are shown.
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ABCB1 p.Arg741Cys 25456855:265:17
status: NEW266 (D) The amount of cross-linked (X-linked) R741C/C-half relative to total R741C/C-half P-gp was determined before ( ) or after (+) treatment with dithiothreitol (DTT).
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ABCB1 p.Arg741Cys 25456855:266:42
status: NEWX
ABCB1 p.Arg741Cys 25456855:266:73
status: NEW269 The 170 kDa protein corresponded in size to R741C/C-half cross-linked to mature A80C/ N-half protein.
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ABCB1 p.Arg741Cys 25456855:269:44
status: NEW272 The results show that R741C/C-half can spontaneously cross-link to A80C/N-half in the endoplasmic reticulum but cross-linking is inhibited by tariquidar.
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ABCB1 p.Arg741Cys 25456855:272:22
status: NEW274 Discussion We found that tariquidar was different from other drug substrates and inhibitors because it blocked A80C/R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:274:116
status: NEW293 How does tariquidar prevent formation of an open conformation in P-gp to block A80C/R741C cross-linking at the cell surface?
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ABCB1 p.Arg741Cys 25456855:293:84
status: NEW294 One reason that tariquidar might block A80C/R741C cross-linking is that it was found to bind very tightly to P-gp at the cell surface [20].
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ABCB1 p.Arg741Cys 25456855:294:44
status: NEW297 Tight binding of tariquidar might influence A80C/R741C cross-linking because modeling studies suggest that the tariquidar binding site lies close to residues at the extracellular ends of TM segments 1 and 7 [49].
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ABCB1 p.Arg741Cys 25456855:297:49
status: NEW299 Occupation of the tariquidar-binding site may block movement between ECL1 and ECL4 required for A80C/R741C cross-linking.
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ABCB1 p.Arg741Cys 25456855:299:101
status: NEW319 The positions of A80C and R741C are indicated as filled balls.
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ABCB1 p.Arg741Cys 25456855:319:26
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
Comments [show]
None has been submitted yet.
No. Sentence Comment
65 Membranes were prepared and samples were incubated at 0 &#b0;C (A80C/R741C, A259C/W803C, I299C/F770C, and A266C/ F1086C) or 20 &#b0;C (L175C/N820C, C431/L1176C, and L521C/C1074) for 10 min in the presence or absence of 0.5 Mapping the P-glycoprotein Tariquidar-binding Site 29390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 49ߦDECEMBER 4, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 11, mM copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 òe;M (for TM segment cysteine mutants) or 1 òe;M tariquidar (for the ICL and NBD cysteine mutants).
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ABCB1 p.Arg741Cys 26507655:65:69
status: NEW69 The reaction mixtures were then subjected to SDS-PAGE (6.5% (w/v) polyacrylamide gels and 7% gels for mutant A80C/R741C) and immunoblot analysis with a rabbit polyclonal antibody against P-gp.
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ABCB1 p.Arg741Cys 26507655:69:114
status: NEW