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)

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys158-Ile159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein, Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).
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PMID:Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L. 155 16

Peptide aldehyde transition state analogue inhibitors of serine and cysteine proteases have been used to selectively inhibit proteases for which prior evidence supports a role in tumor cell metastasis. These enzymes include cathepsin B, urokinase plasminogen activator (PA), and thrombin. The inhibition constants of the peptidyl aldehyde inhibitors show that they are highly selective for a particular targeted serine or cysteine protease. The inhibitors are introduced by i.p. injection or by miniosmotic pumps into syngeneic C57BL/6 mice also given injections of B16-F10 melanoma cells, and the number of metastatic foci in the lung was determined. While the injection protocol gave an initially high but changing in vivo concentration of inhibitor over time, the minipump implant gave a constant steady state concentration of inhibitor over 5-7 days. Minipump infusion of leupeptin (acetylleucylleucylargininal), a strong inhibitor of cathepsin B at a steady state plasma concentration 1000-fold greater than its Ki(cathepsin B), gave no significant decrease in lung colonization by the B16 tumor cells. Ep475, a stoichiometric irreversible peptide inhibitor of cathepsin B-like proteases, also did not significantly inhibit metastatic foci formation. Introduction of selective inhibitors of urokinase PA, tert-butyloxycarbonylglutamylglycyl-argininal and H-glutamylglycylargininal at concentrations near its Ki, produced no significant decrease in mouse lung colonization. The selective thrombin inhibitor D-phenylalanylprolylargininal infused to a steady state concentration 100-fold greater than its Ki dramatically increased B16 melanoma colonization of mouse lung. The results indicate that neither secreted cathepsin B-like nor urokinase PA have roles in B16 colonization of mouse lung, while thrombin may have a role in preventing metastasis. These experiments do not eliminate roles for a cathepsin B-like enzyme or urokinase PA in the initial steps of the metastatic process.
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PMID:Selective inhibition of proteolytic enzymes in an in vivo mouse model for experimental metastasis. 308 87

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.
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PMID:Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. 943 34

A new hypothesis for activation of the contact system of plasma proteolysis (i.e., the plasma kallikrein/kinin system) is presented. Kininogens have a multiprotein receptor on endothelial cells which consists of at least cytokeratin 1, urokinase plasminogen activator receptor, and gC1qR. When contact proteins (high molecular weight kininogen followed by prekallikrein) assemble on the kininogen receptor on endothelial cells, an endothelial cell membrane cysteine protease is expressed to activate prekallikrein to kallikrein. On endothelial cells, prekallikrein activation is independent of factor XIIa activation. Activation of prekallikrein on endothelial cells results in kallikrein cleaving its receptor high molecular weight kininogen to liberate bradykinin. Bradykinin liberation stimulates release of tissue-type plasminogen activator from endothelial cells. Kallikrein formation also results in kinetically favorable pro-urokinase activation on endothelial cells with subsequent plasminogen activation. In addition to stimulating cellular fibrinolysis, kininogens contribute to the constitutive anticoagulant nature of the intravascular compartment. Kininogens block calpain's participation in forming the heterodimeric complex of platelet integrin alpha IIb beta 3. Kininogens also block thrombin from binding to the thrombin receptor(s) on platelets. Last, kininogens prevent thrombin from cleaving protease activated receptor 1 after arginine41. These combined data indicate a biologic system for activation of the plasma kallikrein/kinin system and physiologic consequences as result of this activation.
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PMID:Plasma contact activation: a revised hypothesis. 983 May 13

Fusobacterium nucleatum subsp. nucleatum has been associated with a variety of oral and nonoral infections such as periodontitis, pericarditis, bone infections, and brain abscesses. Several studies have shown the role of plasmin, a plasma serine protease, in increasing the invasive capacity of microorganisms. In this study, we investigated the binding of human plasminogen to F. nucleatum subsp. nucleatum, and its subsequent activation into plasmin. Plasminogen-binding activity of bacterial cells was demonstrated by a solid-phase dot blot assay using an anti-plasminogen antibody. The binding activity was heat resistant and involved cell-surface lysine residues since it was abolished in the presence of the lysine analog epsilon-aminocaproic acid. Activation of plasminogen-coated bacteria occurred following incubation with either streptokinase, urokinase-type plasminogen activator (u-PA), or a Porphyromonas gingivalis culture supernatant. In the case of the P. gingivalis culture supernatant, a cysteine protease was likely involved in the activation. The plasmin activity generated on the cell surface of F. nucleatum subsp. nucleatum could be inhibited by aprotinin. Activation of plasminogen by u-PA was greatly enhanced when plasminogen was bound to bacteria rather than in a free soluble form. u-PA-activated plasminogen-coated F. nucleatum subsp. nucleatum was found to degrade fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue inhibitor of metalloproteinase-1 was also degraded by the plasmin activity generated on the bacterial cells. This study suggests a possible role for plasminogen, which is present in affected periodontal sites, in promoting tissue destruction and invasion by nonproteolytic bacteria such as F. nucleatum subsp. nucleatum.
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PMID:Acquisition of plasmin activity by Fusobacterium nucleatum subsp. nucleatum and potential contribution to tissue destruction during periodontitis. 1056 61

The cysteine protease cathepsin B is upregulated in a variety of tumors, particularly at the invasive edges. Cathepsin B can degrade extracellular matrix proteins, such as collagen IV and laminin, and can activate the precursor form of urokinase plasminogen activator (uPA), perhaps thereby initiating an extracellular proteolytic cascade. Recently, we demonstrated that procathepsin B interacts with the annexin II heterotetramer (AIIt) on the surface of tumor cells. AIIt had previously been shown to interact with the serine proteases: plasminogen/plasmin and tissue-type plasminogen activator (tPA). The AIIt binding site for cathepsin B differs from that for either plasminogen/plasmin or tPA. AIIt also interacts with extracellular matrix proteins, e.g., collagen I and tenascin-C, forming a structural link between the tumor cell surface and the extracellular matrix. Interestingly, cathepsin B, plasminogen/plasmin, t-PA and tenascin-C have all been linked to tumor development. We speculate that colocalization through AIIt of proteases and their substrates on the tumor cell surface may facilitate: (1) activation of precursor forms of proteases and initiation of proteolytic cascades; and (2) selective degradation of extracellular matrix proteins. The recruitment of proteases to specific regions on the cell surface, regions where potential substrates are also bound, could well function as a 'proteolytic center' to enhance tumor cell detachment, invasion and motility.
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PMID:Cell surface complex of cathepsin B/annexin II tetramer in malignant progression. 1070 59

Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly and reassembly of intercellular junctions suggesting germ cell movement is composed of intermittent phases of junction disassembly and reassembly. A study was performed to correlate the expression of junctional-complex components (such as zonula occludens-1 [ZO-1], a tight-junction component protein) and nonjunctional complex components (such as urokinase-type plasminogen activator [uPA], a serine protease; cathepsin L, a cysteine protease; alpha2-macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cysteine protease inhibitor) at the time when inter-Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expression of nonjunctional component genes also correlates with the formation of inter-Sertoli tight junctions in vitro. This is part of an effort to understand the physiologic elements of germ cell movement in the epithelium. Sertoli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of apoptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 x 10(6) cells/cm2 for up to 7 days on bicameral culture units coated with Matrigel (Collaborative Research) and were assessed by morphologic analysis and agarose gel electrophoresis. It was noted that many of the Sertoli cells cultured at 3 x 10(6) cells/cm2 underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 x 10(6) cells/cm2 illustrating the Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO-1 increased significantly between days 2 and 3 prior to the establishment of inter-Sertoli tight junctions assessed by transepithelial resistance measurement (TER), which illustrates that ZO-1 can be used as a marker to monitor this cellular event. More interestingly, there was also a transient increase in the expression of uPA and cathepsin L between days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 x 10(4) cells/cm2), when a confluent monolayer of cells could not form, there were no changes in the expression of either ZO-1, uPA, or cathepsin L throughout the 7-day culture period. These results show that the establishment of specialized junctions, such as tight junctions between Sertoli cells in vitro, may require the participation of both junctional and nonjunctional complex components.
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PMID:Changes in the expression of junctional and nonjunctional complex component genes when inter-sertoli tight junctions are formed in vitro. 1071 17

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.
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PMID:A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site. 1091 10

The SspB cysteine protease of Staphylococcus aureus is expressed in an operon, flanked by the sspA serine protease, and sspC, encoding a 12.9-kDa protein of unknown function. SspB was expressed as a 40-kDa prepropeptide pSspB, which did not undergo autocatalytic maturation. Activity of pSspB was reduced compared with 22-kDa mature SspB, but it was equivalent to mature SspB after incubation with SspA, which specifically removed the pSspB N-terminal propeptide. SspC abrogated the activity of pSspB when incubated in a 1:1 complex but had no effect on SspA or papain. Activity of the pSspB.SspC complex was restored when incubated with SspA, and SspC was cleaved by SspA but not pSspB. Thus, SspC maintains pSspB as an inert zymogen, and SspA is required for removal of the propeptide and inactivation of SspC. Like the papain protease family, SspB cleaved substrates with a hydrophobic amino acid at P2 but had a strong preference for arginine at P1. It did not cleave casein, serum albumin, IgG, or IgA, but it promoted detachment of cultured keratinocytes and cleaved fibronectin and fibrinogen at sites recognized by urokinase plasminogen activator and plasmin, respectively. It also processed high molecular weight kininogen in a manner resembling plasma kallikrein. Thus, SspB exhibits a novel maturation mechanism and mimics the specificity of plasma serine proteases.
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PMID:Identification of a novel maturation mechanism and restricted substrate specificity for the SspB cysteine protease of Staphylococcus aureus. 1220 24

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.
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PMID:Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems. 1295 26


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