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 has accumulated that urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1) and receptor (uPAR) are involved in tumor invasion and metastasis. We analyzed the DNA sequences encoding these factors to see if they are altered in the ovarian cancer cell lines OV-MZ-6, OV-MZ-19, and OVCAR-3. In the uPA-encoding cDNA derived from OV-MZ-6 cells (but not in the uPA-cDNA from OVCAR-3 and OV-MZ-19), a so-far unknown mutation was identified in codon 121, resulting in a proline to leucine exchange. This exchange creates an AluI restriction site making restriction fragment length polymorphism (RFLP) analyses possible. Previously published PAI-1 sequences pointed to a variation of amino acid 15 of the PAI-1 signal sequence representing either threonine or alanine, which was confirmed in the present study. The uPAR cDNAs of all three cell lines encoded the published wild-type sequence. In order to elucidate the possible role of the Pro121Leu exchange in uPA and the Ala/Thr variants in the signal sequence of PAI-1 in the development and/or progression of human ovarian cancer, we studied the presence of these mutants or variants in a series of 22 ovarian cancer tissues. In addition to the wild-type sequence, the Pro121Leu exchange in the uPA sequence was detected in 10 out of 22 tumor tissues; 11 tumors carried exclusively the Pro121 allele; in one case exclusively the Leu121 allele was detected. In 18/22 tumors, triplet 15 in the signal sequence of PAI-1 encoded alanine, four DNAs contained both the Ala and the Thr allele. Furthermore, we analyzed another known common single-base-pair insertion/deletion polymorphism (ins/del allele) found in the promoter region of the PAI-1 gene and thought to be of functional importance in regulating PAI-1 gene expression. The PAI-1 ins-allele was found in 3/22, the del-allele in 6/22 and both alleles in 13/22 ovarian cancer tissues. In genomic DNA isolated from peripheral blood of 23 healthy donors, we observed similar allele frequencies of the three polymorphisms as found in the 22 ovarian carcinomas. Taken together, these results suggest that the polymorphisms observed in the uPA and PAI-1 genes may not be linked to ovarian cancer.
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PMID:Mutational analysis of the genes encoding urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in advanced ovarian cancer. 919 91

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.
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PMID:Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. 926 Nov 79

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
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PMID:Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor. 940 75

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.
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PMID:Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation. 952 80

Staphylokinase (Sak), a 16-kDa bacterial protein, forms a 1:1 stoichiometric complex with the serine proteinase domain of human plasmin, which in turn converts other plasminogen molecules into plasmin. To identify amino acid residues critical for generating the Sak:plasmin activator complex, alanine-scanning mutagenesis was performed on phage-displayed micro-plasminogen (microPlg). Substitution of Arg719 with Ala [microPlg(R719A)] disrupted complex formation, although the sensitivity of phage-displayed microPlg(R719A) to activation by urokinase and the amidolytic activity of the micro-plasmin derivative [microPli(R719A)] remained unaffected. Likewise, the soluble microPlg(R719A) molecule did not generate a functional activator complex with Sak, whereas quantitative activation into plasmin was obtained upon incubation with either urokinase or the Sak:plasmin complex. Real-time biospecific affinity measurements revealed that the Arg --> Ala substitution at position 719 increased the equilibrium dissociation constant between microPlg(R719A) and Sak from 46 nM to 1 microM, primarily by reducing the association rate constant. Arg719 has recently also been implied in the functional complex formation between human plasmin and streptokinase [Dawson, K. M., Marshall, J. M., Raper, R. H., Gilbert, R. J., and Ponting, C. P. (1994) Biochemistry 33, 12042-12047.], suggesting that both bacterial cofactors may share common structural and/or mechanistic aspects for plasminogen activation.
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PMID:Arginine 719 in human plasminogen mediates formation of the staphylokinase:plasmin activator complex. 957 54

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.
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PMID:Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide. 963 68

The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin's broad inhibitory specificity and the role of the secondary binding site. Site-directed and region-specific mutagenesis were preformed at ecotin's primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113). Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding. A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer. The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease. By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed. The contrast in the Ki values of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin's secondary binding site in recognizing these homologous serine proteases. Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes.
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PMID:Ecotin: a serine protease inhibitor with two distinct and interacting binding sites. 964 73

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
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PMID:Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1. 969 48

The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val-->Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80+/-9% (mean+/-S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 86+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degreesC, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52+/-5.2%, 55+/-8.2% and 46+/-4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86+/-1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.
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PMID:Characterization of plasminogen activator inhibitor 1 mutants containing the P13 to P10 region of ovalbumin or antithrombin III: evidence that the P13 residue contributes significantly to the active to substrate transition. 974 34

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.
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PMID:Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. 975 30


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