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)

Urokinase receptors are distributed on surfaces of many cell types where they are thought to focus plasminogen-dependent proteolysis important to migration and tissue remodeling to the immediate pericellular space. In addition to its well characterized role in proteolysis, urokinase receptor binding per se promotes the adhesiveness of leukemic cell lines exposed to differentiating cytokines in vitro. We sought to determine if a serum or matrix component is involved in urokinase-dependent adhesion. We now report that cytokine-stimulated human myelomonocytic cells express a divalent cation- and Arg-Gly-Asp-independent high affinity receptor for urea-purified vitronectin (Kd < 10 nM). Soluble native vitronectin does not effectively bind to the receptor, while cellular adhesion was noted to both urea-purified and native vitronectin when adsorbed to plastic. The activity of this receptor is tightly coupled to urokinase receptor occupancy. Urokinase receptor binding thus induces selective and reversible cellular adhesion to the matrix form of vitronectin. Because transfer of vitronectin-bound plasminogen activator inhibitor type 1 to urokinase promotes rapid turnover of receptor-bound enzyme, these results illuminate a novel binding cycle by which urokinase receptor occupancy coordinately regulates cellular adhesiveness and pericellular proteolysis.
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PMID:Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy. 751 82

Porcine granulosa cells cultured in serum-free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite-2000, an integrin-binding synthetic peptide containing RGD (Arg-Gly-Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin beta 1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F-actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin beta 1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite-2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature commitment of cells towards luteinization rather than completion of follicular preovulatory differentiation.
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PMID:RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro. 754 12

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.
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PMID:Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin. 774 76

The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC). At least five structurally autonomous regions of this three-domain protein have been found to exist. Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease. The latter undergoes three unfolding transitions in its enzymatically active form. Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps. These are attributed to the denaturation of the two major subdomains of the protease structure. One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C. This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded. The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein. These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins.
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PMID:Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains. 813 Feb 9

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

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.
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PMID:Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases. 834 6

Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which reversibly associates with the well characterized neurotrophin NGF-beta. In this study, we demonstrated that NGF-gamma cleaves recombinant single chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or further digestion of tcu-PA by NGF-gamma, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF-gamma resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 +/- 0.6) x 10(-2) s-1 and 2.3 +/- 0.4 microM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-gamma was 1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 for the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-gamma, as determined by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl coumarin. By activating scu-PA, NGF-gamma may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-beta activities which involve cellular migration and/or extracellular matrix remodeling.
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PMID:Nerve growth factor-gamma activates soluble and receptor-bound single chain urokinase-type plasminogen activator. 839 59

A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees C), ranges from 0.29 x 10(7) M-1s-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) to 1.08 x 10(7) M-1s-1 (tcr-[KuPA-->K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA-->K1HPgK4HPg]uPA) and 2.4 (r-[KuPA-->K2HPgK3HPg]uPA) relative to that of wtr-uPA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen. 851 11

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

The effect of fibrin on angiogenesis in vitro was investigated using an experimental model of tube formation by bovine capillary endothelial cells (BCEs) in type I collagen gel. One milligram per milliliter of fibrin added into type I collagen gel significantly increased the length of the tubular structures formed by BCEs in the gel by about 180% compared with type I collagen only. The facilitating effect of fibrin on tube formation by BCEs was inhibited by either anti-basic fibroblast growth factor (bFGF) IgG (25 micrograms/ml) or anti-urokinase type plasminogen activator (uPA) IgG (10 micrograms/ml) added to the gel and culture medium, but not by anti-tissue type plasminogen activator (uPA) IgG (10 micrograms/ml) added to the gel and culture medium, but not by anti-tissue type plasminogen activator (10 micrograms/ml) or non-immune IgG. The Arg-Gly-Asp (RGD) containing peptides (100 micrograms/ml) added to the culture medium also suppressed tube formation by BCEs in fibrin-containing type I collagen gel, but not in type I collagen gel. These results suggest that the increased release of bFGF and uPA by BCEs therefore plays a role in the angiogenic effect of fibrin in vitro, and the angiogenic effect of fibrin is mediated by the RGD sequence in fibrin, probably via the function of integrin receptor of the BCEs.
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PMID:Effects of fibrin on the angiogenesis in vitro of bovine endothelial cells in collagen gel. 858 91


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