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)

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
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PMID:Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. 218 Apr 85

The cDNA encoding human urokinase (UK) has been isolated from a cDNA library prepared from human normal fibroblast (WI38) cells, which had been stimulated by endothelial cell growth factor and heparin. This cDNA was sequenced and found to contain a few silent substitutions, thus encoding the same amino acids as deduced from the published genomic sequence of UK. After modification, the cDNA of UK was inserted into a transient expression vector and used to transfect COS-1 cells. The recombinant UK protein (rUK) in the serum-free medium of transfected COS-1 cells was characterized by biochemical and functional assays. These studies indicated that rUK from COS-1 cells is glycosylated, enzymatically active, and very similar to native single-chain plasminogen activator (scuPA). Therefore, such rUK can be a convenient source of scuPA for any further studies.
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PMID:Isolation of a human cDNA of urokinase and its expression in COS-1 cells. 323 71

The urokinase receptor (uPAR) is linked to plasma membranes through a glycosylphosphatidylinositol (GPI) anchor. It has been posited that the GPI anchor facilitates clearance of uPAR-bound complexes between two chain urokinase (tcuPA) and plasminogen activator inhibitor type 1 (PAI-1) by the alpha 2-macroglobulin receptor (alpha 2MR) which permits re-expression of unoccupied uPA receptors on the cell surface. To test this hypothesis we compared internalization and degradation of 125I-labeled tcuPA-PAI-1 by COS cells expressing either transfected wild-type, GPI-linked uPAR (uPAR/GPI), or a chimeric receptor composed of the extracellular domains of uPAR linked to the transmembrane and cytosolic domains of the alpha chain (p55 subunit) of the interleukin-2 receptor (uPAR/IL-2R alpha). The kinetics of binding, internalization and degradation of tcuPA-PAI-1 by COS cells expressing each form of uPAR were virtually identical. However, internalization of complexes by uPAR/IL-2R alpha was more susceptible to inhibition by recombinant soluble 39-kDa alpha 2MR-associated protein (RAP) which competes for binding of tcuPA-PAI-1 complexes to alpha 2MR (p < 0.001), and the internalization was accompanied by a greater reduction in the number of surface uPAR/IL-2R alpha, than uPAR/GPI (p < 0.05). These studies indicate that the rate of internalization of tcuPA-PAI-1 is governed primarily by the extracellular domains of uPAR, whereas the GPI anchor may facilitate internalization of complexes and re-expression of uPAR when binding sites on alpha 2MR are limiting.
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PMID:Endocytosis of urokinase-plasminogen activator inhibitor type 1 complexes bound to a chimeric transmembrane urokinase receptor. 751 Jun 79

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.
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PMID:Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library. 753 22

Complex between urokinase and its type-1 inhibitor (uPA-PAI-1) may, when bound to the urokinase receptor (uPAR), be endocytosed by an ensuing binding of the complex to the multiligand receptors alpha (2)-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330). We have found that phorbol esters regulate endocytosis of uPA-PAI-1 differently in different cell lines. In COS-1 cells, expressing uPAR and high levels of alpha 2MR/LRP under basal conditions, phorbol esters cause a time-dependent decrease in endocytosis concomitantly with a parallel down-regulation of alpha 2MR/LRP expression. An up-regulation of uPAR expression was also observed. General endocytosis via the clathrin-coated pit pathway was not affected by PMA treatment, as judged from measurements of transferrin endocytosis. In LLC-PK1 cells, expressing alpha 2MR/LRP but not uPAR under basal conditions, phorbol esters transiently increase endocytosis in parallel with a transient induction of uPAR expression, while there was virtually no change in alpha 2MR/LRP expression. Differential regulation of endocytosis therefore seems to be caused by differential regulation of the receptors, with either the alpha 2MR/LRP-level (in COS)-1 cells) or the uPAR-level (in LLC-PK1 cells) being rate-limiting.
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PMID:Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of alpha 2-macroglobulin receptor and urokinase receptor expression. 766 84

Affinity selection of a 15-mer random peptide library displayed on bacteriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially and alternately selecting the peptide library on COS-7 monkey kidney cells and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regions of the selected bacteriophage were synthesized and tested in a urokinase receptor binding assay, where they competed with the labeled N-terminal fragment of urokinase with IC50 values ranging from 10 nM to 10 microM. All of the isolated peptides were linear and showed two relatively short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFXXYLW, neither of which is found in urokinase or its receptor. Competition experiments demonstrated that the most potent peptide, clone 20, prevented binding of bacteriophage displaying the urokinase receptor binding sequence (urokinase residues 13-32). In addition, this peptide blocked other apparently unrelated receptor binding bacteriophage, suggesting overlapping receptor interaction sites for all of these sequences. These results provide a demonstration of bacteriophage display identifying peptide ligands for a receptor expressed on cells and yield leads for the development of urokinase receptor antagonists.
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PMID:High-affinity urokinase receptor antagonists identified with bacteriophage peptide display. 804 58

We have used Chinese hamster ovary (CHO) cells to express high levels of human prourokinase gene cDNA with recourse to construction of good expression vector, the improvement of transfection technique and gene coamplification. First, we constructed expression plasmid pMG10102 by placing pro-UK cDNA under the control of SR alpha promoter/SV40 polyadenylation signals and expressed it transiently in COS-7 cells. Expression level was about 5 times higher than with SV40 early promoter. Linear plasmids pMG10102 and pSV2-dhfr were then cotransfected into CHO-dhfr cells by calcium phosphorate coprecipitation and cells were cultured in selective medium. Twenty transformants expressing pro-UK were picked, the range of expression levels was 12.5-100IU/10(6) cells/day. When subjected to stepwise selection of methotrexate (MTX), the stable cell lines were obtained that secreted up to 400-500IU/10(6) cells/day. Western blot analysis showed that molecular weight of secreted recombinant pro-UK was the same as that of natural pro-UK, which is 52 kDa, and more than 60% of expression production was single chain urokinase (rscUK) without protease inhibitor in medium.
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PMID:High level expression of human prourokinase cDNA in Chinese hamster ovary cells. 804 46

Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.
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PMID:Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed mutagenesis of a putative nuclear translocation sequence. 814 56

Recombinant f1 phage particles containing cDNA of the human urokinase type-plasminogen activator (u-PA) under the control of the simian virus 40 (SV40) early promoter were used for transfection of monkey COS-7 cells using a DEAE dextran method. The fibrinolytic activity of u-PA was detected in the culture medium of the 10(5) cells transfected with the phage particles containing single-stranded (ss) DNA of more than 0.2 ng. This finding will lead us to develop a simple and efficient method for expression cloning using mammalian cells.
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PMID:Recombinant f1 phage particles can transfect monkey COS-7 cells by DEAE dextran method. 848 95

Cell-specific activation by follicle-stimulating hormone and its intracellular mediator, cAMP, of the human urokinase promoter in mouse Sertoli cells requires overlapping purine-rich and GC-rich sequences between -54 and -42 from the transcriptional start site. We have previously shown that binding of unidentified nuclear factors to these sequences is induced by cAMP stimulation, and that sequences from the enhancerless SV40 replication origin can interfere with the binding, whereas consensus Sp1 binding sites are ineffective. We now show that sequences within the SV40 origin able to compete for the formation of cAMP-induced DNA-protein complexes in Sertoli cell nuclear extracts are binding sites for the SV40 large T antigen. Large T antigen expressed in COS cells binds the cAMP-responsive sequences of the human urokinase gene and transactivates the proximal promoter, thus mimicking the effect of nuclear factors induced by cAMP in Sertoli cells. We show that Egr-1 is one of the factors present in cAMP-induced DNA-protein complexes formed between the human urokinase promoter and Sertoli cell nuclear extracts. However, Egr-1 levels are similar in unstimulated and cAMP-treated Sertoli cells, suggesting that this factor interacts with a different GC-box binding factor, that we have previously shown to be strongly induced by cAMP treatment of Sertoli cells. We propose that SV40 large T antigen in COS cells can mimick the action of heterodimers formed in cAMP stimulated Sertoli cells between Egr-1 and a cell specific cAMP-induced GC-box binding factor.
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PMID:The same sequence mediates activation of the human urokinase promoter by cAMP in mouse Sertoli cells and by SV40 large T antigen in COS cells. 873 76


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