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)

Down-regulation of oncogene expression is one of the hallmarks of the process whereby transformed cells are forced into differentiation and/or growth arrest by potent inducers and therefore can represent an interim end point in cancer treatment. The differentiation inducer sodium butyrate (NaB) arrested growth of N.1 ovarian carcinoma cells and repressed expression of cyclin D1/prad1 and the invasiveness-related protease plasminogen activator-urokinase (plau). This was accompanied by the acquisition of a differentiated morphology, all of which characteristics were maintained as long as N.1 cells were exposed to the inducer. In accordance with a differentiated phenotype was the finding that fibronectin expression was increased significantly. Recently, it was shown that NaB represses the transcription factor c-myc by blocking Ca2+ signals and modulating serine threonine kinase activity. We wanted to investigate NaB-mediated interference on signals contributing to the expression on prad1, plau and growth arrest-specific 6 (gas6). Protein kinase A (PKA) inactivation de-repressed prad1 and plau transcript levels. NaB had onlygeneral but no specific influence on PKA-modulated prad1 and plau expression however. Protein kinase C activation up-regulated plau transcript levels, but not that of prad1. Prad1 expression seemed to depend on Ca2+-triggered signals. Constitutive plau expression was insensitive to additional Ca2+-mediated signals, but it became responsive upon NaB treatment.
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PMID:Genes related to growth and invasiveness are repressed by sodium butyrate in ovarian carcinoma cells. 859 56

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.
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PMID:Enhancement of catalytic activities of serine proteases by tripeptides compounds. 863 1

Extensive tissue remodeling occurs in survivors of acute lung injury, leading to nearly normal histology and physiology in the majority of individuals, whereas others suffer significant impairment due to the development of pulmonary fibrosis. Alveolar epithelial cells play a central role in the repair process. They are strategically located to directly participate in the solubilization of intraalveolar fibrin deposits, and have the capacity to promote fibrinolysis. We have previously reported that interleukin-1 beta (IL-1 beta), an important inflammatory mediator in acute lung injury, upregulates urokinase-type plasminogen activator expression by human A549 cells (1). In this work, we show that IL-1 beta increases cell-surface plasmin generation, mediated in part by increased expression of urokinase receptor (u-PAR). Northern blot analyses demonstrated that IL-1 beta rapidly induces accumulation of u-PAR messenger RNA (mRNA) in a dose-dependent fashion, and that this effect is blocked by actinomycin. The IL-1 beta-mediated increase in u-PAR mRNA is inhibited by: (1) the relatively specific protein kinase C (PKC) inhibitors 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) and calphostin C; and (2) prolonged pretreatment of cells with phorbol myristate acetate (PMA), suggesting that PKC is an important component of the signaling pathway. Okadaic acid, an inhibitor of serine/threonine phosphatases, markedly potentiates the effect of IL-1 beta on u-PAR mRNA levels. In contrast, dexamethasone, in concentrations as low as 10(-8) M, completely blocks the IL-1 beta-mediated increase in u-PAR mRNA. Half-life experiments show that dexamethasone has no effect on u-PAR mRNA stability. Aldosterone, at concentrations in which it binds primarily to the mineralocorticoid receptor, has no effect on u-PAR expression, suggesting that the glucocorticoid effect is due to a transrepressive mechanism. In summary, IL-1 beta increases cell-surface plasmin generation in A549 cells by coordinately upregulating urokinase and u-PAR expression. Transcriptional activation of the u-PAR gene involves PKC-dependent mechanisms, and glucocorticoid suppression is probably due to interactions between the glucocorticoid receptor and another transcriptional activating system such as activator protein-1 (AP-1) and/or nuclear factor-kB (NF-kB).
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PMID:Induction of urokinase-type plasminogen activator receptor by IL-1 beta. 919 70

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

beta2-Glycoprotein I (beta2GPI) is a highly glycosylated plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids. The most relevant physiological role of beta2GPI is supposed to be the regulation of the function of anionic phospholipids like cardiolipin (CL). beta2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V). In the previous study, we found that factor Xa can produce the nicked form by cleaving Lys 317-Thr 318, using recombinant human domain V (r-Domain V). However, the reaction was extremely slow. In the present paper, we found that plasmin can produce the nicked form of domain V, using recombinant domain V (r-Domain V) and beta2GPI from human plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, r-Domain V was rapidly cleaved into a nicked form by plasmin, very slowly by factor Xa, but not by thrombin, tissue-type plasminogen activator, urokinase, and tissue factor/factor VIIa. The cleavage site of r-Domain V and beta2GPI by plasmin was proved to be Lys 317-Thr 318 by amino acid sequence analysis of the digest and of the C-terminal peptide isolated by high-performance liquid chromatography. The cleavage was completely inhibited by plasmin inhibitor (alpha2PI). The nicked form was demonstrated to show reduced affinity for CL with a dissociation constant of one order of magnitude larger than that of the intact beta2GPI. To determine whether the specific cleavage of beta2GPI by plasmin can occur also in plasma, human plasma was first acid-treated to inactivate alpha2PI and then incubated with urokinase. About 12% of beta2GPI in plasma was nicked when alpha2PI activity decreased to 80%. The nicked form was not generated in plasminogen-depleted plasma. These results suggest that plasmin can produce the nicked form of beta2GPI with the reduced ability to bind phospholipids in vivo.
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PMID:Plasmin can reduce the function of human beta2 glycoprotein I by cleaving domain V into a nicked form. 959 64

The activation status of the ras pathway was studied in eight ovarian tumor cell lines. Three biochemical parameters indicative of ras activation were tested: (a) the ratio of the ras-GTP:ras-GDP complex; (b) the activity of mitogen-activated protein kinases p42/p44; and (c) ets-2 phosphorylation at position threonine 72, a mitogen-activated protein kinase phosphorylation site in vivo. Four of the ovarian tumor cell lines had an activated ras pathway by these three parameters, whereas only one of these contained a mutated ras gene. In addition, ras/ets-2 responsive genes such as the urokinase plasminogen activator (uPA) were activated in these four cell lines. Transient transfection assays indicated that the compound ets-AP1 oncogene responsive enhancer present in the uPA gene was the target of ras signaling in ovarian tumor cells and that the combination of activated ras and ets-2 could superactivate the uPA enhancer element. Coexpression of the dominant-negative ras-Asn17 cDNA gene abrogated activity of this uPA element in ovarian tumor cells. These data indicate that ets-2 is a nuclear target of ras action in ovarian tumor cell lines and that ras signaling pathways may be activated in ovarian cancer by mechanisms independent of direct genetic damage to ras genes.
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PMID:Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines. 960 74

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.
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PMID:Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms. 1050 32

A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-.) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser(441)-Val(442) peptide bond in human plasmin(ogen), with additional cleavage of the Lys(78)-Val(79) and Pro(447)-Val(448) peptide bonds, and a secondary cleavage at Lys(615)-Val(616). Thereby, plasminogen is converted into an angiostatin-like fragment containing kringles 1-4 (K1-4) and miniplasminogen (kringle 5 and the serine proteinase domain). The purified K1-4 fragment showed a comparable cytotoxicity toward endothelial cells as the elastase-derived K1-3 fragment (12.7% versus 10.6% at a concentration of 10 microg/mL). Plasminogen, bound to monocytoid THP-1 cells, was also cleaved by the 24k-endopeptidase, resulting in generation of an angiostatin-like fragment and in a decreased capacity to generate cell-associated plasmin following activation by urokinase. The 24k-endopeptidase was not efficiently neutralized by specific inhibitors against the serine, cysteine, aspartic, or matrix metalloproteinase classes of enzymes. In human plasma or serum, however, it induced only very limited plasminogen degradation, apparently due to neutralization of its activity by alpha(2)-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.
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PMID:Specific proteolysis of human plasminogen by a 24 kDa endopeptidase from a novel Chryseobacterium Sp. 1063 Oct 10

The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase-stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase-Akt-JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival.
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PMID:ets-2 is a target for an akt (Protein kinase B)/jun N-terminal kinase signaling pathway in macrophages of motheaten-viable mutant mice. 1102 73

The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
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PMID:[Single-chain urokinase-type plasminogen activator (scu-PA) purification by immuno-affinity chromatography]. 1219 74


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