Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
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PMID:Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. 796 84

HGF is secreted by mesenchymal cells and regulates motogenesis, mitogenesis, and morphogenesis of epithelial and endothelial cells. HGF is a heterodimer of two glycosylated chains, alpha and beta, bound together by a disulfide bond. The molecule is synthesized as single chain precursor devoid of biological activity (pro-HGF). The critical step in pro-HGF activation is a proteolytic cleavage generating the two chain form. This step occurs in the extracellular environment, and is catalyzed by urokinase. Two alternative transcripts originate two HGF variants. One bears a deletion of five amino acids in the alpha chain, and has the same properties of the full-size protein. The other one contains only the first portion of the alpha chain (two kringle HGF). Two kringle HGF binds the HGF receptor, triggers its tyrosine kinase activity and behaves as a partial agonist, inducing motogenesis but not mitogenesis in target cells. The HGF receptor is the tyrosine kinase encoded by the c-MET pro-oncogene, a tyrosine kinase receptor. This molecule is an heterodimer of an extracellular alpha chain disulfide linked to a transmembrane beta chain. The cytoplasmic portion of the beta chain contains the catalytic domain and critical sites for the regulation of its kinase activity. In the C-terminal tail, a bidentate motif containing two tyrosines associates the transducers responsible for HGF signalling.
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PMID:Hepatocyte growth factor and its receptor, the tyrosine kinase encoded by the c-MET proto-oncogene. 798 17

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
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PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.
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PMID:Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity. 820 90

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.
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PMID:Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line. 840 7

Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum-free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.
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PMID:Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells. 844 89

A conjugate of annexin V and the B-chain of urokinase was prepared and its fibrinolytic properties were studied. First, a mutant of annexin V was constructed with an N-terminal extension of six amino acids (Met-Ala-Cys-Asp-His-Ser) and with Cys316 mutated to Ser; this molecule was expressed in Escherichia coli. The urokinase B-chain was prepared by limited reduction of the interchain disulfide bond between the A- and B-chains of urokinase. These two molecules were then then connected by a disulfide bond and purified to yield a 1:1 stoichiometric conjugate. The conjugate had the same catalytic activity as urokinase against a synthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activating activity. The conjugate showed the same binding affinity for phosphatidylserine-containing membranes as annexin V. The in vitro fibrinolytic activity of the conjugates on clots prepared from platelet-rich plasma was comparable to that of urokinase. However, the conjugate showed 3-4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasma clearance rate as urokinase or B-chain. These results show that annexin V is a useful agent for targeting plasminogen activators to phospholipid-containing thrombi.
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PMID:Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi. 854 74

Kaposi's sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposi's-sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion-derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte-growth-factor receptor (c-MET). Moreover, elevated levels of transforming growth factor beta 1 (TGF beta 1) mRNA were found in lesions of human sporadic Kaposi's sarcoma and in lesion-derived spindle cells which also over-express urokinase. Since HGF, TGF beta 1 and urokinase are all involved in capillary-vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposi's sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.
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PMID:Over-expression of hepatocyte growth factor in human Kaposi's sarcoma. 856 12

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. Although HGF/SF is synthesized by mesenchymal cells and acts predominantly on epithelial cells, we have recently demonstrated that human sarcoma cell lines often inappropriately express high levels of Met and respond mitogenically to HGF/SF. In the present report we show that HGF/SF-Met signalling in the human leiomyosarcoma cell line SK-LMS-1 enhances its in vivo tumorigenicity, an effect for which the mitogenicity of this signalling pathway is likely to play a role. In addition, we found that HGF/SF-Met signalling dramatically induces the in vitro invasiveness and in vivo metastatic potential of these cells. We have studied the molecular basis by which HGFSF-Met signalling mediates the invasive phenotype. A strong correlation has previously been demonstrated between the activation of the urokinase plasminogen activator (uPA) proteolysis network and the acquisition of the invasive-metastatic phenotype, and we show here that HGF/SF-Met signalling significantly increases the protein levels of both uPA and its cellular receptor in SK-LMS-1 cells. This results in elevated levels of cell-associated uPA and enhanced plasmin-generating ability by these cells. These studies couple HGF/SF-Met signalling to the activation of proteases that mediate dissolution of the extracellular matrix-basement membrane, and important property for cellular invasion-metastasis.
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PMID:Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-met signalling in human cells concomitant with induction of the urokinase proteolysis network. 862 56

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.
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PMID:Regulation of the urokinase-type plasminogen activator gene by the oncogene Tpr-Met involves GRB2. 903 78


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