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: UNIPROT:O95477 (membrane-bound)
29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein alpha (FAPalpha), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAPalpha primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinity-labeled by [3H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that seprase, FAPalpha, and DPPIV are related serine integral membrane proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.
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PMID:Identification of the 170-kDa melanoma membrane-bound gelatinase (seprase) as a serine integral membrane protease. 906 13

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.
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PMID:Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation. 908 58

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.
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PMID:Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA). 915 9

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as CD26, is a membrane-bound serine protease that cleaves off aminoterminal dipeptides from peptides with a penultimate proline (or, at a much slower rate, a penultimate alanine). Recently, we synthesized and characterized a number of dipeptide-derived diphenylphosphonates. Out of the resulting series of slow-binding irreversible inhibitors of DPP IV, diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate hydrochloride (Pro-Pro-diphenylphosphonate or Prodipine) was selected for further study. We investigated the in vivo applicability of Prodipine. Male rabbits weighing 3-4 kg received a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity had decreased to less than 20% of the preinjection value and remained unchanged during a 24-hr observation period. In a next step, we aimed to study (i) the dose dependency and (ii) the duration of the effect after a single intravenous dose of Prodipine. A profound and long-lasting inhibition of plasma DPP IV activity was observed in the treated animals (1, 5 or 10 mg). It took 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery. Systemic treatment with Prodipine not only led to inhibition of plasma DPP IV activity but also decreased tissue DPP IV activity in circulating mononuclear cells, kidney cortex, thymus, spleen, lung, and liver. No differences in activities of the related peptidases aminopeptidase P (APP, EC 3.4.11.9), prolyl oligopeptidase (PO, EC 3.4.21.26), or aminopeptidase M (mAAP, EC 3.4.11.2) were detected between Prodipine-treated and control rabbits. The in vivo applicability of this chemically stable, irreversible inhibitor of DPP IV opens new possibilities, not only to further unravel the biological functions of this intriguing ectopeptidase, but also to explore this enzyme as a new target in various fields of pharmacological research.
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PMID:In vivo inhibition of dipeptidyl peptidase IV activity by pro-pro-diphenyl-phosphonate (Prodipine). 929 64

Human coagulation factor IX (FIX) is a serine protease which binds to a negatively charged phospholipid surface in the presence of Ca ions (Ca2+). FIX two-dimensional (2-D) crystals were obtained by the lipid layer crystallisation technique under near physiological conditions. The 2-D projection map of the protein was calculated to a resolution of 3 nm using electron crystallographic analysis. The structural organisation of membrane-bound FIX is discussed and compared with the known X-ray crystallographic data.
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PMID:Structural determination of lipid-bound human blood coagulation factor IX. 960 16

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.
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PMID:Protease in normal human epidermal keratinocytes. 970 64

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.
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PMID:Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes. 976 22

It has been shown that erythrocyte membrane proteins become susceptible to degradation by membrane-bound serine protease activity after oxidative modification of the membranes (M. Beppu, M. Inoue, T. Ishikawa, K. Kikugawa, Biochim. Biophys. Acta 1196 (1994) 81-87). The aim of the present study was to clarify the presence of the serine protease in oxidized erythrocyte membranes and to characterize the selectivity of the enzyme to oxidized proteins. Human erythrocytes were oxidized in vitro with xanthine/xanthine oxidase/Fe(III) and oxidized membranes isolated. Proteolytic activity of the membranes toward spectrin obtained from oxidized membranes and bovine serum albumin oxidized with H2O2/horseradish peroxidase was increased by membrane oxidation, and the degradability of the substrates was increased by substrate oxidation. The proteolytic activity was inhibited by the serine protease inhibitor diisopropyl fluorophosphate (DFP). The 72 kDa and 80 kDa proteins in the membranes were labeled by [3H]DFP when detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and subsequent fluorography. The 72 kDa protein was found to be a serine enzyme, acetylcholine esterase. The 80 kDa protein appeared to be responsible for the degradation of oxidatively damaged proteins. The 80 kDa protein was loosely bound to membranes and readily solubilized into a 0.1% NP-40 detergent solution. The presence of the same 80 kDa protease in intact erythrocyte cytosol was suggested. The increased serine protease activity in oxidized membranes can result from the increased adherence of the cytosolic 80 kDa serine protease to the membranes due to oxidation.
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PMID:Characterization of membrane-bound serine protease related to degradation of oxidatively damaged erythrocyte membrane proteins. 981 51

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-psi[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.
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PMID:Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics. 982 45

CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis.
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PMID:CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration. 1005 Aug 80


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