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:3.4.21.73 (urokinase-type plasminogen activator)
10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.
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PMID:Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid. 1074 93

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, efficiently inhibits tumor cell invasion and metastasis. We examined the effect of UTI on urokinase-type plasminogen activator (uPA) expression in ovarian cancer cell lines, HOC-I and HRA. By Northern blot, Western blot, ELISA, and zymographic analyses, we demonstrated that UTI inhibited the expression of uPA mRNA and protein in these cells in a time- and dose-dependent manner, independent of whether induction was triggered by phorbol ester. Monoclonal antibody 4G12, which inhibits UTI binding to the cells, produced a dose-dependent abrogation in UTI-mediated down-regulation of uPA expression. These data suggest that UTI significantly down-regulates tumor cell uPA mRNA expression and protein secretion, and that UTI binding to the cells is necessary to exert the UTI's action.
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PMID:Suppression of urokinase-type plasminogen activator expression from human ovarian cancer cells by urinary trypsin inhibitor. 1101 22

Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.
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PMID:Adenoviral gene transfer of a u-PA receptor-binding plasmin inhibitor and green fluorescent protein: inhibition of migration and visualization of expression. 1101 72

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.
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PMID:Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C- and MEK/ERK/c-Jun-dependent signaling pathways. 1105 91

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(45)). Here we characterize binding properties of UTI-BPs.UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP(40) and UTI-BP(45) bind (125)I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40). Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP(45). Analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45). In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.
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PMID:Characterization of binding properties of urinary trypsin inhibitor to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8. 1127 81

Injury-induced neointimal development results from migration and proliferation of vascular smooth muscle cells (SMC). Cell migration requires controlled proteolytic degradation of extracellular matrix surrounding the cell. Plasmin is a major contributor to this process by degrading various matrix proteins directly, or indirectly by activating matrix metalloproteinases. This makes it an attractive target for inhibition by gene transfer. An adenoviral vector, Ad.ATF.BPTI, was constructed encoding a hybrid protein, which consists of the aminoterminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) linked to bovine pancreas trypsin inhibitor (BPTI), a potent inhibitor of plasmin. This hybrid protein binds to the u-PA receptor, thereby inhibiting plasmin activity at the cell surface, and was found to be a potent inhibitor of cell migration in vitro. Local infection with Ad.ATF.BPTI of balloon-injured rat carotid artery resulted in detectable expression of ATF.BPTI mRNA and protein in the vessel wall. Morphometric analysis of arterial cross-sections revealed that delivery of Ad.ATF.BPTI to the carotid artery wall at the time of balloon injury inhibited neointima formation by 53% (P < 0.01) at 14 days and 19% (P = NS) at 28 days after injury when compared with control vector-infected arteries. Intima/media ratios were decreased by 60% (P < 0.01) and 35% (P < 0.05) at 14 and 28 days, respectively, when compared with control vector-infected arteries. Furthermore, a small but significant increase in medial area was found in the Ad.ATF.BPTI-treated arteries at 28 days (P < 0.05). These results show that local infection of the vessel wall with Ad.ATF.BPTI reduces neointima formation, presumably by inhibiting SMC migration, thereby offering a novel therapeutic approach to inhibiting neointima development.
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PMID:In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI. 1131 20

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.
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PMID:Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins. 1134 88

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.
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PMID:Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix. 1158 17

Bikunin (bik, also known as urinary trypsin inhibitor [UTI]), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) in order to evaluate the effect on expression of urokinase-type plasminogen activator (uPA). Preincubation of the cells with bik reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. We next asked whether the mechanism of inhibition of uPA expression by bik is due to interference with MAP kinase, since PMA could also activate a signaling pathway involving MEK/ERK/c-Jun-dependent uPA expression. When cells were preincubated with bik, we could detect suppression of phosphorylation of these proteins, demonstrating that bik markedly suppresses the cell motility possibly through negative regulation of MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes. To clarify the role of bik on tumor metastasis, HRA cells were transfected with an expression vector harboring a cDNA encoding for human bik. Transfection of HRA with the bik cDNA resulted in five variants stably expressing functional bik and significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells. Animals with bik* transfectants induced reduced peritoneal dissemination and long term survival. These results suggest that transfection with the bik gene induces the suppression of tumor cell invasion and peritoneal dissemination, and can prolong survival. This pre-clinical animal model offers the possibility to explore gene therapy as a new treatment modality for ovarian cancer.
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PMID:Suppression of urokinase expression and tumor metastasis by bikunin overexpression [mini-review]. 1177 42

Investigations determined that the cell matrix-associated prekallikrein (PK) activator is prolylcarboxypeptidase. PK activation on human umbilical vein endothelial cell (HUVEC) matrix is inhibited by antipain (IC(50)=50 microM) but not anti-factor XIIa antibody, 3 mM benzamidine, 5 mM iodoacetic acid or iodoacetamide, or 3 mM N-ethylmaleimide. Corn trypsin inhibitor (IC(50)=100 nM) or Fmoc-aminoacylpyrrolidine-2-nitrile (IC(50)=100 microM) blocks matrix-associated PK activation. Angiotensin II (IC(50)=100 microM) or bradykinin (IC(50)=3 mM), but not angiotensin 1-7 or bradykinin 1-5, inhibits matrix-associated PK activation. ECV304 cell matrix PK activator also is blocked by 100 microM angiotensin II, 1 microM corn trypsin inhibitor, and 50 microM antipain, but not angiotensin 1-7. 1 mM angiotensin II or 300 microM Fmoc-aminoacylpyrrolidine-2-nitrile indirectly blocks plasminogen activation by inhibiting kallikrein formation for single chain urokinase activation. On immunoblot, prolylcarboxypeptidase antigen is associated with HUVEC matrix. These studies indicate that prolylcarboxypeptidase is the matrix PK activator.
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PMID:Identification of prolylcarboxypeptidase as the cell matrix-associated prekallikrein activator. 1212 26


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