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)

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion in vitro and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.
 ...
PMID:Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin. 943 20

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.
 ...
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

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.
 ...
PMID:Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells. 959 42

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.
 ...
PMID:Ecotin: a serine protease inhibitor with two distinct and interacting binding sites. 964 73

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.
 ...
PMID:Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator. 964 77

Metastasis is a characteristic and fatal feature of human malignancies. Its regulation is therefore of the utmost significance to clinicians. The present study was undertaken to determine whether a legume-derived protease inhibitor (PI) of trypsin/chymotrypsin, the field bean PI (FBPI), also has plasmin inhibitory activity and can inhibit pulmonary metastasis of B16F10 melanoma cells systemically injected into BDF1 mice. Two approaches to the problem were made. In the first, the melanoma cells were exposed to two different concentrations of the FBPI prior to their inoculation into animals. In the second, the mice were treated intraperitoneally with FBPI at a dose of 100 mg/kg body weight once daily for 10 days, the treatment being started soon after the systemic injection of the tumour cells. The study revealed that both modes of FBPI treatment could effectively block lung cell metastasis by the melanoma cells and that FBPI has plasmin blocking activity. Since urokinase type plasminogen activator and plasmin are known to play significant roles in tumour cell metastasis, the dose-dependent inhibitory effect of FBPI with antiplasmin activity on tumour cell metastasis suggests that its antimetastatogenic action is probably mediated through its plasmin inhibitory action.
 ...
PMID:The field bean protease inhibitor has the potential to suppress B16F10 melanoma cell lung metastasis in mice. 971 30

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.
 ...
PMID:Serine proteases in rodent hippocampus. 972 24

Human kallikrein 2 (hK2) is a serine protease expressed predominantly in the prostate which has 80% homology to prostate-specific antigen (PSA). hK2 is an active trypsin-like protease which has been shown by immuno-histochemical staining to be more highly expressed in prostate carcinoma than in benign prostate tissue. Unlike PSA, hK2 activates pro-PSA , pro-hK2 and the zymogen form of urokinase-type plasminogen activator (uPA), an extracellular protease correlated with prostate cancer and metastasis. We show here that hK2 rapidly forms a complex with plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of uPA in tissues. In addition, hK2 inactivated 6 to 7 mol of PAI-1 by cleavage at Arg346-Met347 for every mole of hK2-PAI-1 complex formed. In contrast with hK2, PSA neither complexed with nor inactivated PAI-1. PAI-1 inhibited hK2 comparably with protein C inhibitor (PCI) and at least 20 times more rapidly than alpha1-anti-chymotrypsin (ACT). N-Terminal sequencing shows that hK2 forms a covalent complex with PAI-1, PCI and ACT after cleavage at Arg346-Met347, Arg354-Ser355 and Leu358-Ser359, respectively. During complex formation, hK2 inactivated PAI-1 but did not inactivate ACT or PCI. Our current results suggest that the increased hK2 expression in prostate cancer tissues could influence cancer biology not only by activation of uPA but also by inactivation of its primary inhibitor, PAI-1.
 ...
PMID:Prostatic human kallikrein 2 inactivates and complexes with plasminogen activator inhibitor-1. 1020 59

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) alphavbeta3, beta1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.
 ...
PMID:Src-dependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. 1039 32

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.
 ...
PMID:Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. 1050 Jan 22


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