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)

Protease nexin-I (PN-I) is representative of a newly described class of serine protease inhibitors secreted by human fibroblasts, the protease nexins. Protease nexins form covalent complexes with their target proteases, subsequently binding to cells via specific receptors. PN-I preferentially binds thrombin, urokinase, trypsin, and plasmin, and its binding to thrombin is accelerated by heparin. We have previously described the production of a polyclonal antibody against PN-I which is able to block the binding of PN-I X proteinase complexes to cells and will immunoprecipitate metabolically labeled PN-I. Anti-PN-I was used to investigate the biosynthesis and regulation of PN-I in human fibroblasts. Unlabeled PN-I could compete for the binding of metabolically labeled PN-I to anti-PN-I, as shown by the elimination of the 43-kDa band representing PN-I on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs. Excision of this 43-kDa band from gels, followed by amino-terminal sequencing, showed a homogeneous protein that is homologous with that described by Scott et al. (Scott, R. W., Bergman, B. L., Bajpai, A., Hersh, R. T., Rodriguez, H., Jones, B. N., Barreda, C., Watts, S., and Baker, J. B. (1985) J. Biol. Chem. 260, 7029-7034). An analysis of the biosynthesis of the PN-I revealed that a lower Mr precursor exists intracellularly. This apparent rough endoplasmic reticulum form appears as a doublet on sodium dodecyl sulfate gels, as does mature PN-I. The PN-I precursor was also sensitive to endoglycosidase H, suggesting that it contains N-linked carbohydrates of the high mannose form. Mature PN-I is not sensitive to endoglycosidase H, but does contain 3 kDa of N-linked carbohydrate. PN-I appears to be constitutively secreted by fibroblasts. PN-I levels in conditioned media reach a steady state within 48 h, although PN-I synthesis maintains a constant rate. This steady state is due to the continuous uptake of PN-I from medium, presumably through a specific receptor.
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PMID:Biosynthesis of protease nexin-I. 377 29

The host reaction is an important factor in the biological behavior of cancers. In human colon adenocarcinoma, stromal cells and some cancer cells express the urokinase receptor (uPAR), a molecule involved in the regulation of extracellular proteolysis. The present study reveals the identity of uPAR-expressing cell types and the subcellular localization of this molecule by immunoelectron microscopy in colon cancer. uPAR-positive cells were most abundant at the invasive margin of colon cancer and were identified as macrophages, fibroblasts, neutrophilic and eosinophilic granulocytes, endothelial cells and cancer cells. Of these, the most numerous were macrophages with uPAR detected along the plasma membrane, in accordance with its function in plasminogen activation on the cell surface. Fibroblasts were labeled in the lumen of rough endoplasmic reticulum, indicating its intracellular synthesis. Some granulocytes and endothelial cells expressed immunoreactivity along the plasma membrane. uPAR-positive cancer cells were stained along the plasma membrane and in rough endoplasmic reticulum. These findings suggested that a variety of non-malignant host cells play an important role in the plasmin-mediated breakdown of the extracellular matrix at the invasive margin.
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PMID:Expression of urokinase receptor in various stromal-cell populations in human colon cancer: immunoelectron microscopical analysis. 755 16

Direct retrovirus-mediated hepatic gene transfer results in permanent gene expression; however, gene transfer requires surgical hepatectomy (to stimulate cell division) and has been inefficient. We recently used recombinant adenovirus vectors that transiently expressed urokinase from mouse hepatocytes to induce hepatocellular regeneration in place of a partial hepatectomy. The adenovirus method allowed for five-fold more efficient retrovirus transduction in vivo compared to the conventional partial hepatectomy approach. The major problem with the urokinase-mediated hepatic regeneration was the transient secretion of urokinase into the bloodstream that led to hypocoagulation. To circumvent this side-effect, the urokinase protein was modified by adding amino-terminal and carboxy-terminal endoplasmic reticulum retention signals. The recombinant urokinase molecules expressed from adenoviral vectors remained in hepatocytes, were enzymatically active, and resulted in similar rates of hepatic regeneration as found with the secreted urokinase. Modified urokinase-mediated liver regeneration was equally capable of allowing retrovirus-mediated gene transfer in vivo. Thus, the method of direct retrovirus transduction of hepatocytes becomes clinically relevant as the technology becomes safer.
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PMID:A modified urokinase plasminogen activator induces liver regeneration without bleeding. 757 15

Variations in glycosylation exist among urokinase plasminogen activator receptors (u-PARs) from different cell types. We have studied the functional role of N-linked carbohydrate within the ligand-binding domain of u-PAR. Treatment with glycosidases demonstrated that all the N-linked carbohydrates on u-PAR are complex-type oligosaccharides. Substitution of a single Asn (Asn52) to Gln by means of site-directed mutagenesis led to an active receptor mutant with a ligand-binding domain devoid of carbohydrate. The cellular distribution, the glycosyl-phosphatidylinositol anchoring, and the conformational stability after solubilization were unaffected by this single substitution. However, ligand binding analysis demonstrated a 4- 5-fold decrease in affinity as compared with the wild type receptor. Two different strategies were used in order to obtain a u-PAR type completely devoid of N-linked carbohydrates. 1) Tunicamycin treatment of wild type u-PAR-expressing cells. 2) Mutation of all glycosylation sites (Hu-PARN5-mut). In neither case, unglycosylated receptors with ligand binding activity were identified. However, immunofluorescence studies demonstrated that the Hu-PARN5-mut was retained inside the cells in the endoplasmic reticulum. The same result was found for Hu-PARN4-mut, where only the glycosylation sites outside the binding domain were mutated. These results demonstrate that some extent of glycosylation of u-PAR is necessary for cellular transport and for molecular maturation events leading to ligand binding activity. Glycosylation of the binding domain per se affects only the affinity of the receptor. The positive modulation of the Asn52 carbohydrate side chain on ligand affinity suggests that the u-PAR glycosylation variants observed in various cell types may have different functional roles.
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PMID:N-linked glycosylation of the ligand-binding domain of the human urokinase receptor contributes to the affinity for its ligand. 838 83

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.
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PMID:Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries. 935 35

Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter.
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PMID:Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor. 1072 33

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.
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PMID:Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain. 1263 3

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal. The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.
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PMID:C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha. 1487 36

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.
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PMID:Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha. 1620 May 4

In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.
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PMID:Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway. 1749 12


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