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)

During human blood clotting, alpha2-antiplasmin (alpha2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha2AP and a specific epsilon-lysyl group in each of the alpha-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified alpha2AP, which lacked plasmin-inhibitory activity, competed effectively with native alpha2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant alpha2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant alpha2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant alpha2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native alpha2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of alpha2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native alpha2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant alpha2AP displayed full plasmin inhibitory activity, while mutant alpha2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type alpha2AP. When our mutant alpha2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant alpha2AP augmented lysis by competing with native alpha2AP for FXIIIa-catalyzed incorporation into fibrin.
 ...
PMID:Characterization of wild-type and mutant alpha2-antiplasmins: fibrinolysis enhancement by reactive site mutant. 1038 9

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.
 ...
PMID:Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis. 1060 68

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.
 ...
PMID:Specific proteolysis of human plasminogen by a 24 kDa endopeptidase from a novel Chryseobacterium Sp. 1063 Oct 10

Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1'-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1'(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.
 ...
PMID:Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. 1083 93

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.
 ...
PMID:A region in domain II of the urokinase receptor required for urokinase binding. 1086 23

Matriptase is an epithelial-derived, integral membrane serine protease. The enzyme was initially isolated from human breast cancer cells and has been implicated in breast cancer invasion and metastasis. In the current study, using active matriptase isolated from human milk, we demonstrate that matriptase is able to cleave various synthetic substrates with arginine or lysine as their P1 sites and prefers small side chain amino acids, such as Ala and Gly, at P2 sites. For the most reactive substrates, N-tert-butoxycarbonyl (N-t-Boc)-gamma-benzyl-Glu-Ala-Arg-7-amino-4-methylcoumarin (AMC) and N-t-Boc-Gln-Ala-Arg-AMC, the K(m) values were determined to be 3. 81 and 4.89 microm, respectively. We further demonstrated that matriptase can convert hepatocyte growth factor/scattering factor to its active form, which can induce scatter of Madin-Darby canine kidney epithelial cells and can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells. In addition, we noted that matriptase can activate urokinase plasminogen activator but has no affect on plasminogen. These results suggest that matriptase could act as an epithelial, upstream membrane activator to recruit and activate stromal-derived downstream effectors important for extracellular matrix degradation and epithelial migration, two major events of tissue remodeling, cancer invasion, and metastasis.
 ...
PMID:Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. 1096 9

By using PCR and DNA recombination, two fusion genes of humanized mouse anti-human fibrin scFv and low molecular weight single chain urokinase (Scu-PA-32K) was constructed. The difference of these two fusion genes lay in the linker between two moieties, one was (Ala)3 and another was (Gly4Ser)3. These two fusion genes were both overexpressed in E. coli with the expression level at 30%. Both expression products showed the activity of binding antigen D-Dimer and activating plasminogen after the denaturation and renaturation, but under general refolding conditions, the one with linker (Gly4Ser)3 showed better effect in the renaturation of fusion protein.
 ...
PMID:[The construction and expression of two humanized scFv-urokinase fusion genes]. 1105 31

The urokinase plasminogen activator receptor (uPAR) plays an important role in the migration of leukocytes. It occurs as a membrane-bound form that contains a glycosylphosphatidylinositol (GPI) anchor and also as a soluble form (suPAR) that lacks the GPI anchor. Recently, a sequence of amino acids, SRSRYLE, within the receptor has been found to become unmasked on uPA binding or chymotrypsin cleavage. Exposure of the epitope results in the activation of p56/p59(hck) kinase and chemotaxis of myelomonocytic cells. Using an epitope-tagged suPAR molecule, we found that both three-domain and two-domain suPAR promote the adhesion of differentiated THP-1 cells to fibronectin and vitronectin, indicating that suPAR can modify cell adhesion as well as cell migration. In addition, we found that the amino acid sequence RYLE, within the chemotactic peptide, is conserved across species and that alanine substitution of Tyr 92 decreased the ability of the peptide to activate p56/59(hck).
 ...
PMID:Soluble urokinase receptor promotes cell adhesion and requires tyrosine-92 for activation of p56/59(hck). 1109 55

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.
 ...
PMID:Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3). 1116 24

Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.
 ...
PMID:Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. 1130 69


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>