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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human MDR1 gene product, P-glycoprotein (Pgp), a tandemly duplicated molecule containing two putative ATP- and perhaps two drug-binding sites, is responsible for multidrug resistance in tumors. In this report, we characterized the effects of trypsinization of Pgp on its ATPase function. Incubation of Pgp-containing membranes with trypsin at a ratio of 1000:1 (w/w) resulted in a gradual increase in the basal- and the drug-stimulated ATPase activities of Pgp in a time-dependent manner. The maximal basal-, verapamil-, and vinblastine-stimulated ATPase activities of the trypsinized Pgp were approximately 1.8-, 1.5-, and 1.75-fold higher than the activities of the native Pgp, respectively. Increased basal- and drug-stimulated ATPase activities of the Pgp were also observed when the ratio of membrane protein to trypsin in the incubation mixtures was raised to 10:1 (w/w). Immunoblotting analysis of Pgp tryptic digests using Pgp-specific NH(2)11, C219, and C494 antibodies together revealed the degradation of full-length Pgp and formation of at least eight peptides migrating in the 36-60 kDa range. Immunoprecipitation reactions using NH(2)11 and C494 antibodies have suggested that the peptides originating from the NH(2) half of Pgp are in strong association with the COOH half of the peptide. These findings suggest that while Pgp fragments together exhibit the ATPase functional characteristics, Pgp possesses a cleavage activation site or region, and its cleavage leads to the activation of basal ATPase function of Pgp.
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PMID:Activation of the human P-glycoprotein ATPase by trypsin. 1072 37

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.
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PMID:Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1. 1074 57

Limited trypsin digestion was used to monitor nucleotide-induced conformational changes in wild-type P-glycoprotein (Pgp) as well as in nucleotide binding domain (NBD) Pgp mutants. Purified and reconstituted wild-type or mutant mouse Mdr3 Pgps were preincubated with different hydrolyzable or nonhydrolyzable nucleotides, followed by limited proteolytic cleavage at different trypsin:protein ratios. The Pgp tryptic digestion products were separated by SDS-PAGE followed by immunodetection with the mouse monoclonal anti-Pgp antibody C219, which recognizes a conserved epitope (VVQE/AALD) in each half of the protein. Different trypsin digestion patterns were observed for wild-type Pgp incubated with MgCl(2) alone, MgADP, MgAMP.PNP, MgATP, and MgATP + vanadate. A unique trypsin digestion profile suggestive of enhanced resistance to trypsin was observed under conditions of vanadate-induced trapping of nucleotides (MgATP + vanadate). The trypsin sensitivity profiles of Pgp mutants bearing either single or double mutations in Walker A (K429R, K1072R) and Walker B (D551N, D1196N) sequence signatures of NBD1 and NBD2 were analyzed under conditions of vanadate-induced trapping of nucleotides. The proteolytic cleavage pattern observed for the double mutants K429R/K1072R and D551N/D1196N, and for the single mutants K429R, K1072R, and D1196N were similar and clearly distinct from wild-type Pgp under the same conditions. This is consistent with the absence of ATP hydrolysis and of vanadate-induced trapping of 8-azido-ADP previously reported for these mutants [Urbatsch et al. (1998) Biochemistry 37, 4592-4602]. Interestingly, the trypsin digestion profiles observed under vanadate-induced trapping for the D551N and D1196N mutants were quite different, with the D551N mutant showing a profile resembling that seen for wild-type Pgp. The different sensitivity profiles of Pgp mutants bearing mutations at the homologous residue in NBD1 (D551N) and NBD2 (D1196N) suggest possible structural and functional differences between the two sites.
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PMID:Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity. 1075 6

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.
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PMID:The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites. 1082 82

Desloratadine is a new, selective, H(1)-receptor antagonist that also has anti-inflammatory activity. In vitro studies have shown that desloratadine inhibits the release or generation of multiple inflammatory mediators, including IL-4, IL-6, IL-8, IL-13, PGD(2), leukotriene C(4), tryptase, histamine, and the TNF-alpha-induced chemokine RANTES. Desloratadine also inhibits the induction of cell adhesion molecules, plateletactivating factor-induced eosinophil chemotaxis, TNF-alpha-induced eosinophil adhesion, and spontaneous and phorbol myristate acetate-induced superoxide generation in vitro. In animals desloratadine had no effect on the central nervous, cardiovascular, renal, or gastrointestinal systems. Desloratadine is rapidly absorbed, has dose-proportional pharmacokinetics, and has a half-life of 27 hours. The absorption of desloratadine is not affected by food, and the metabolism and elimination are not significantly affected by the subject's age, race, or sex. There are no clinically relevant interactions between desloratadine and erythromycin, ketoconazole, or grapefruit juice. Desloratadine is not a significant substrate of the P-glycoprotein transport system. Once daily administration of desloratadine rapidly reduces the nasal and nonnasal symptoms of seasonal allergic rhinitis, including congestion. In patients with seasonal allergic rhinitis and concomitant asthma, desloratadine treatment was also associated with significant reductions in total asthma symptom score and use of inhaled beta(2)-agonists. Use of desloratadine in patients with chronic idiopathic urticaria was associated with significant reductions in pruritus, number of hives, size of the largest hive, and interference with sleep and daily activities. Clinical experience in over 2300 patients has shown that the adverse event profile of desloratadine is similar to that of placebo; desloratadine has no clinically relevant effects on electrocardiographic parameters, does not impair wakefulness or psychomotor performance, and does not exacerbate the psychomotor impairment associated with alcohol use.
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PMID:Desloratadine: A new, nonsedating, oral antihistamine. 1129 78

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [(3)H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [(3)H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.
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PMID:Characterization of the dexniguldipine binding site in the multidrug resistance-related transport protein P-glycoprotein by photoaffinity labeling and mass spectrometry. 1202 98

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.
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PMID:Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function. 1205 98

BMS-262084 is a potent and selective beta-lactam tryptase inhibitor with therapeutic potential for treating asthma. The oral bioavailability of BMS-262084 was low in rats (4% at a dose of 0.5 mg/kg) due to poor absorption. BMS-262084 was excreted mainly unchanged in the urine, suggesting minimal metabolism in rats. The objective of this study was to investigate the mechanisms of oral absorption of BMS-262084 in rats. Modulation of intestinal tight junctions, binding to trypsin, and involvement of the intestinal dipeptide transport system and P-glycoprotein (P-gp) in the absorption of BMS-262084 were examined. Coadministration of BMS-262084 with SQ-29852, a substrate of the intestinal dipeptide transport system, did not change the oral absorption of BMS-262084. An increase in the dose of BMS-262084 from 0.5 to 50 mg/kg resulted in a 3.7-fold increase in its oral absorption. Inulin absorption was enhanced upon coadministration with BMS-262084, suggesting the opening of tight junctions in the intestinal epithelium. Coadministration of aprotinin, a trypsin inhibitor, increased the oral absorption of BMS-262084 several fold. In vitro, using Caco-2 cells, BMS-262084 appeared to be a P-gp substrate, with an efflux ratio of 14. These results suggest that absorption of BMS-262084 is mediated by several concurrent mechanisms. At higher doses of BMS-262084, increased absorption may be primarily due to opening of tight junctions in the intestinal epithelium and consequent absorption via the paracellular pathway, while at lower doses, binding to trypsin may contribute to limiting its absorption. P-gp efflux may also play a role in influencing the absorption of BMS-262084. The intestinal dipeptide transporter system does not appear to be involved in the absorption of BMS-262084.
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PMID:Multiple pathways are involved in the oral absorption of BMS-262084, a tryptase inhibitor, in rats: role of paracellular transport, binding to trypsin, and P-glycoprotein efflux. 1579 9

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.
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PMID:Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein. 1650 85

The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
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PMID:Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates. 1662 45


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