ABCMdb

ABCB1 p.Gly268Val

Predicted by SNAP2:	A: D (53%), C: N (57%), D: N (57%), E: D (75%), F: D (71%), H: D (71%), I: D (71%), K: D (75%), L: D (75%), M: D (71%), N: N (93%), P: D (80%), Q: D (66%), R: D (75%), S: N (82%), T: N (53%), V: D (53%), W: D (80%), Y: D (75%),
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] Rescue of DeltaF508 and other misprocessed CFTR mu... Mol Pharm. 2005 Sep-Oct;2(5):407-13. Loo TW, Bartlett MC, Clarke DM
Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound.
Mol Pharm. 2005 Sep-Oct;2(5):407-13., [PMID:16196493]

Abstract [show]
Cystic fibrosis (CF) is most commonly caused by deletion of Phe508 in the cystic fibrosis transmembrane conductance regulator protein (DeltaF508 CFTR). The misfolded DeltaF508 CFTR protein is retained in the endoplasmic reticulum (misprocessed mutant) and is rapidly degraded. Studies on misprocessed mutants of P-glycoprotein (P-gp), a sister protein of CFTR, however, have shown that specific substrates and modulators can act as specific chemical/pharmacological chaperones to rescue the protein. A major goal in CF research is the identification of compounds that can be used at low concentrations to rescue misprocessed CFTR mutants. Here, we show that a novel quinazoline derivative, 4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)piperazin-1-yl]ethyl}qu inazoline (CF(cor)-325), rescued DeltaF508 CFTR. Incubation of BHK cells stably expressing human DeltaF508 CFTR with 1-10 microM CF(cor)-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CF(cor)-325 in either BHK or HEK 293 cells. CF(cor)-325 appeared to be specific for DeltaF508 CFTR because another quinazoline derivative, prazosin, did not rescue the misprocessed CFTR mutants. CF(cor)-325 could also rescue misprocessed mutants of P-gp. The compound was a P-gp inhibitor as it inhibited vinblastine-stimulated ATPase activity. P-gp-mediated vinblastine resistance was also reduced about 10-fold with 300 nM CF(cor)-325. These results show that CF(cor)-325 is a particularly important lead compound for treatment of CF because low concentrations can be used to rescue many misprocessed CFTR mutants.

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149 An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment
150 Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment
164 Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment
165 (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment
170 The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment
147 An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment
148 Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment
162 Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment
163 (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment
168 The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment

[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PMID:18708637]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.

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47 The G268V and L1260A processing mutations were introduced into the L443C(NBD1)/S909C(TMD2) mutant. Login to comment
108 The locations of processing mutations G268V and L1260A that were used to inhibit maturation of P-gp are indicated as squares. Login to comment
136 To test if NBD-TMD2 interactions differ in the mature and immature forms of P-gp, the G268V (26) or L1260A (38) processing mutations were introduced into mutant L443C(NBD1)/ S909C(TMD2) to inhibit its maturation. Login to comment
140 The G268V mutation is located in the second intracellular loop in TMD1 whereas the L1260A mutation is located at the COOH-end of NBD2. Login to comment
159 Mutants G268V/L443C(NBD1)/S909C(TMD2) and L1260A/L443C(NBD1)/S909C(TMD2) were expressed in HEK 293 cells in the presence of no drug (None) or 10 ␮m cyclosporin A (ϩ Cyclo). Login to comment
199 Processing mutations near the NH2- (G268V) or COOH-end (L1260A) of the molecule disrupted cross-linking between Cys-443(NBD1) and Cys-909(TMD2). Login to comment
5 The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/ A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Login to comment

[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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64 Accordingly, the histidine-tagged 150 kDa protein of mutants G268V, G269V, and Y710A were isolated (Fig. 1, lanes 1, 3, and 5). Login to comment
66 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). Login to comment
73 As reported previously (19), the mature enzyme of mutants G268V and G269V had ϳ50 to 75% of the activity of wild-type P-gp. Login to comment
76 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. Login to comment
84 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. Login to comment
88 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. Login to comment
196 Potential strategies would be to identify a pharmacological agent(s) that causes exposure of hypersensitive-protease sites such as R113 (Fig. 8, step 1), that can induce the same alterations as in the processing mutants (e.g., G268V, Fig. 8, step 2), or that blocks protein folding steps as seen with protease inhibitors (Fig. 8, step 3). Login to comment

[hide] Rescue of folding defects in ABC transporters usin... J Bioenerg Biomembr. 2005 Dec;37(6):501-7. Loo TW, Bartlett MC, Clarke DM
Rescue of folding defects in ABC transporters using pharmacological chaperones.
J Bioenerg Biomembr. 2005 Dec;37(6):501-7., [PMID:16691490]

Abstract [show]
The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (DeltaF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in DeltaF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of DeltaF508 CFTR as well as other CFTR processing mutants in the presence of 1 muM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.

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76 The presence of a processing mutation in either half-molecule (N-half (G268V) or COOH-half (A841L)) resulted in the loss of interactions between the half-molecules. Login to comment

[hide] Correction of defective protein kinesis of human P... J Biol Chem. 1997 Jan 10;272(2):709-12. Loo TW, Clarke DM
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem. 1997 Jan 10;272(2):709-12., 1997-01-10 [PMID:8995353]

Abstract [show]
There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

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51 RESULTS Effect of Drug Substrates on Processing of Misfolded Mutants-The effect of substrates and modulators of P-glycoprotein on the biosynthesis of two processing mutants were initially studied; G268V in the NH2-terminal transmembrane domain (16) and ⌬Y490 in the NH2-terminal nucleotide-binding domain (17). Login to comment
52 Mutant G268V is a temperature-insensitive processing mutant, whereas mutant ⌬Y490 contains a deletion at an equivalent position to the ⌬F508 mutation in CFTR. Login to comment
64 In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown). Login to comment
73 Fig. 2A shows that after 4 h in the presence of 15 ␮M cyclosporin A, about 50% of mutant G268V was present as the fully mature (170-kDa) form of the enzyme and that after 24 h, more than 80% of the mutant protein was present in the fully mature form. Login to comment
77 Fig. 2B shows that in the absence of cyclosporin A, the 150-kDa P-glycoprotein of mutant G268V was not processed to the mature enzyme. Login to comment
79 In the presence of cyclosporin A, however, the kinetics of maturation of the P-glycoprotein of mutant G268V was similar to that of wild-type enzyme. Login to comment
102 By contrast, most of the P-glycoprotein of mutants G268V and ⌬Y490 grown without drug substrate were recovered in the flow-through fractions during nickel-chelate chromatography. Login to comment
107 Similarly, drug-stimulated ATPase activity was detected in the mutant G268V after expression in the presence of cyclosporin A. Login to comment
108 The observation that mutant G268V exhibits reduced activity is consistent with previous observations that several glycine to valine mutations in the cytoplasmic loops of P-glycoprotein also alter the substrate specificity of the enzyme (16). Login to comment
110 Time-dependent appearance of the 170-kDa (mature) form of mutant P-glycoprotein-A52 (G268V). Login to comment
111 A, HEK 293 cells were transfected with A52-tagged mutant G268V cDNA and incubated for 24 h at 37 °C. Login to comment
114 B, HEK 293 cells were transfected with wild-type or mutant G268V P-glycoprotein-A52 cDNAs or vector alone (Control). Login to comment
140 Another interesting observation is that misfolded mutants that are temperatureand glycerol-insensitive, such as G251V, G268V, and E707A could also be rescued by these drug substrates when expressed in either HEK 293 or NIH 3T3 cells (data not shown). Login to comment

[hide] Superfolding of the partially unfolded core-glycos... J Biol Chem. 1998 Jun 12;273(24):14671-4. Loo TW, Clarke DM
Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions.
J Biol Chem. 1998 Jun 12;273(24):14671-4., 1998-06-12 [PMID:9614062]

Abstract [show]
Misprocessed mutants of human P-glycoprotein accumulate as core-glycosylated intermediates in the endoplasmic reticulum and are rapidly degraded. Trypsin digestion was used to test for structural differences between mature and core-glycosylated forms of P-glycoprotein. We found that the core-glycosylated wild-type and mutant P-glycoproteins were both 100-fold more sensitive to trypsin compared with the mature form of the wild-type enzyme. This result suggested that the core-glycosylated forms of both wild-type and mutant P-glycoproteins have similar unfolded structures, whereas the mature enzyme is folded into a more compact structure. The core-glycosylated mutant P-glycoproteins could be converted to the mature trypsin-resistant form by synthesis in the presence of drug substrate. Addition of proteasome inhibitor MG-132 to stabilize the core-glycosylated intermediate resulted in the accumulation but not maturation of the mutant protein. Further analysis showed that the second transmembrane domain TMD2 also became more resistant to trypsin digestion only after coexpression with TMD1 in the presence of substrate. Taken together, these results suggest that simply stabilizing the core-glycosylated intermediate is not sufficient to promote maturation of the processing mutants and that drug substrates induce maturation by promoting superfolding of the transmembrane domains.

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52 When mutant G268V was expressed in HEK 293 cells (no drug, lane 1), the major product was a 150-kDa core-glycosylated protein that was sensitive to digestion with endoglycosidase H (data not shown). Login to comment
54 We attempted to purify the core-glycosylated intermediate of mutant G268V to determine whether it was still active. Login to comment
63 As shown in Fig. 1A (no drug), the 150-kDa protein of mutant G268V was quite sensitive to trypsin. Login to comment
74 To test this possibility, we expressed mutant G268V in the presence of a proteasome inhibitor (MG-132). Login to comment
76 As shown in Fig. 1C (lanes 3 and 4), incubation of mutant G268V for 24 h in the presence of MG-132 resulted in accumulation of the core-glycosylated intermediate and a 130-kDa digestion product. Login to comment
78 Increased accumulation of the core-glycosylated intermediate and the 130-kDa digestion product was also seen with the FIG. 1. Protease sensitivity of mutant G268V and wild-type P-glycoproteins. Login to comment
79 Membranes prepared from HEK 293 cells expressing mutant G268V (A) or wild-type (B) P-glycoproteins-A52 grown without (no drug) or with (ϩ drug) 10 ␮M CsA were treated with TPCK-trypsin followed by immunoblot analysis. Login to comment
81 C, HEK 293 cells transfected with wild-type or G268V cDNAs were incubated for 24 h in media containing 5 ␮M MG132 (ϩ) or no inhibitor (-), and equivalent amounts of whole cell extracts were immunoblotted with monoclonal antibody A52. Login to comment
89 We first introduced a processing mutation (G268V) into N-Half-His. Login to comment
90 Unlike the histidine-tagged full-length G268V P-glycoprotein, the mutant (G268V) N-Half-His could be recovered by nickel-chelate chromatography, even when grown with no CsA (Fig. 2A). Login to comment
91 Wild-type or G268V N-Half-His molecules were then coexpressed with the C-Half-A52 molecule. Login to comment
93 As shown in Fig. 2B, C-Half-A52 was not retained on the column when expressed alone or when coexpressed with mutant G268V N-Half- His in the absence of CsA. Login to comment
94 In contrast, CsA promoted interaction between C-Half-A52 and mutant G268V N-Half-His, such that most of the C-Half-A52 was now recovered by nickel-chelate chromatography. Login to comment
111 A, membranes from HEK 293 cells expressing histidine-tagged wild-type or G268V N-half molecules were treated for 24 h with (ϩ) or without (-) 10 ␮M CsA, solubilized with 1% (w/v) n-dodecyl-beta-D-maltoside, and the supernatants were subjected to nickel-chelate chromatography. Login to comment
113 B, detergent-solubilized HEK 293 membranes expressing only C-Half-A52, C-Half-A52, and wild-type N-Half-His, or both C-Half-A52 and G268V N-Half-His grown with (ϩ) or without (-) 10 ␮M CsA were subjected to nickel-chelate chromatography. Login to comment
120 B, wild-type or mutant (G268V) N-Half-A52 was coexpressed with wild-type C-Half-His in the presence of 10 ␮M CsA. Login to comment

[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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114 Accordingly, cells expressing a misprocessed mutant (G268V) with or without an additional R113A mutation were radiolabeled with [35 S]methionine and [35 S]cystine to monitor the fate of the mutant. Login to comment
115 Mutant G268V is a temperature-insensitive processing mutant (8) that yields mature enzyme (170 kDa) only when synthesized in the presence of drug substrates (7). Login to comment
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). Login to comment
118 Mutant G341C appeared to have a greater folding defect than the G268V misprocessed mutant. Login to comment
134 FIG. 4. Effect of R113A mutation on maturation of wild type and mutant G268V P-gp. HEK 293 cells expressing wild type or mutant G268V P-gps-A52 and containing Arg113 or R113A were pulse-labeled for 20 min at 37 °C and then chased with fresh media for the various intervals (hr). Login to comment

[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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96 The I261S, V264S, F267A, G268V, and G269V mutations blocked maturation such that the 150-kDa immature protein was the major product (Fig. 1B). Login to comment

[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.

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163 First, we tested if tariquidar could rescue the G268V processing mutant. Login to comment
164 The G268V mutant was selected because we previously found that it could be rescued with vinblastine, verapamil, or cyclosporine A [44]. Login to comment
174 processing mutations like G268V inhibit P-gp maturation because they trap P-gp in the endoplasmic reticulum as a partially folded immature 150 kDa core-glycosylated protein. Login to comment
177 Mutant G268V containing an A52 epitope tag was transiently expressed in HEK 293 cells in the presence of various concentrations of tariquidar for 18 h. Login to comment
183 The results show that tariquidar acted as a potent pharmacological chaperone to promote maturation of G268V P-gp. Login to comment
184 By comparison, 20-fold higher concentrations of high affinity drug substrates/modulators such as verapamil, vinblastine or cyclosporine A were required to promote maturation of G268V P-gp [44]. Login to comment
199 These concentrations of drug substrates were chosen because they maximally promoted maturation of the G268V mutant [44]. Login to comment
208 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. Login to comment