Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.
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PMID:Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. 938 Jun 80

P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.
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PMID:Conformational changes of P-glycoprotein by nucleotide binding. 939 36

Human breast carcinoma MCF-7/AdrVp cells display a novel multidrug resistance phenotype that is characterized by the overexpression of a 95-kDa membrane glycoprotein (p95) and by marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein MRP. p95 is also highly expressed in multidrug-resistant NCI-H1688 cells derived from a human small cell lung carcinoma. Deglycoslyated p95 from NCI-H1688 cells was isolated by two-dimensional gel electrophoresis and then digested with trypsin. The tryptic peptides were analyzed by mass spectrometry and microsequencing. These analyses identified p95 to be identical to NCA-90, the nonspecific cross-reacting antigen related to the carcinoembryonic antigen (CEA). Further confirmation that p95 is indeed NCA-90 was obtained by Northern and Western blot studies using probes or antibodies specific for p95, NCA-90, or CEA family members. Western blot studies also revealed that CEA itself is overexpressed in MCF-7/AdrVp cells compared to parental MCF-7/W cells. The enforced expression of NCA-90 protein in HeLa cells stably transfected with NCA-90 cDNA did not result in increased resistance of the transfected cells to daunorubicin or a decrease in daunorubicin accumulation in the transfected cells compared to cells transfected only with the expression vector. However, a recent report by H. Kawaharata et al. (Int. J. Cancer, 72: 377-382, 1997) of diminished accumulation, retention, and cytotoxicity of doxorubicin in EJNIH3T3 cells in which enforced expression of CEA was accomplished leaves open the possibility that the overexpression of CEA, possibly in combination with that of NCA-90, could account at least in part for the drug resistant phenotype displayed by MCF-7/AdrVp cells.
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PMID:The 95-kilodalton membrane glycoprotein overexpressed in novel multidrug-resistant breast cancer cells is NCA, the nonspecific cross-reacting antigen of carcinoembryonic antigen. 940 50

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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PMID:Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions. 961 62

Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.
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PMID:Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites. 964 11

P-glycoprotein (Pgp) is a membrane-transport ATPase. It uses energy from ATP hydrolysis to transport pleiotropic cytotoxic drugs from inside to outside of cells. Thus, elevated expression of Pgp in cancer cells causes multidrug resistance. It is now known that the conformational state of Pgp changes during its catalytic cycle. However, how ATP hydrolysis relates to drug binding by Pgp is yet to be determined. In this study, we used limited trypsin digestion of Pgp in isolated inside-out membrane vesicles to investigate the effects of drugs on Pgp conformation and to determine the drug-bound conformational states of Pgp in the catalytic cycle. We found that (a) binding of vinblastine or verapamil alone can cause a conformational change in Pgp, but the change induced by the drug binding is different from that induced by nucleotide binding, (b) there may be at least two binding sites for Pgp substrates, one for drugs such as vinblastine and verapamil and the other for drugs such as colchicine and adriamycin, (c) the conformation of Pgp bound by ATP and vinblastine is different from the conformation bound by either one alone, and (d) the ADP-bound Pgp does not bind vinblastine. Based on these observations and our previous studies, we propose a model for drug binding and transport in the catalytic cycle of Pgp.
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PMID:Dissection of drug-binding-induced conformational changes in P-glycoprotein. 971 79

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.
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PMID:Identification of the cyclosporin-binding site in P-glycoprotein. 992 80

The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.
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PMID:The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface. 1045 47

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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PMID:The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy. 1050 75

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.
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PMID:Both ATP sites of human P-glycoprotein are essential but not symmetric. 1052 34


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