Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have screened six human squamous carcinoma cell lines for their ability to invade connective tissue by using the experimentally modified chorioallantoic membrane of a chick embryo as an in vivo model of invasion. In confirmation of our earlier studies, all the invasive cell lines expressed high levels of surface-bound urokinase type plasminogen activator (uPA). However, some cell lines expressing this activity were not invasive, suggesting that surface uPA, although necessary, was not sufficient. Since in addition to fibronectin, that can be degraded by uPA or plasmin, chorioallantoic membrane connective tissue contains collagen, we examined the profile of collagenases secreted by the various cell lines in search for an activity that would coincide with the invasive phenotype. We found, using gelatin substrate gels, that type IV gelatinase was produced by all six cell types tested, three cell types produced the M(r) 92,000 gelatinase, and three a lower-molecular-weight activity, which we identified by immunoprecipitation with specific antibodies, and by a direct assay of activity, as interstitial collagenase. Only the latter cells were found to be highly invasive. We showed previously that continuous culture in vitro of one of the carcinoma cell lines, HEp3, led to a gradual extinction of their malignant phenotype. To confirm the correlation between invasion and the production of interstitial collagenase, we examined these two functions in cells freshly isolated from a HEp3 tumor and intermittently during passage in vitro. We found that, although the surface uPA activity was slightly diminished in the in vitro grown cultures, it was still within the range of values found in highly malignant cells, suggesting that it is not the reason for the decrease in invasiveness. In contrast, the reduction in interstitial collagenase closely followed the loss of the invasive phenotype; after 30 in vitro passages the cells were almost completely devoid of interstitial collagenase and unable to invade. The decrease in collagenase activity was not the result of an increased tissue inhibitor of metalloproteinases production.
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PMID:Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. 133 82

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

Extracellular matrix metalloproteases are synthesized as proenzymes and are activated by certain physiological agents after secretion into the extracellular space. The identity of these agents and the stimulus that elicits their response in vivo is only recently becoming clear, but a variety of agents or stimuli are capable of activating these metalloproteases in vitro also. Of these, the most well studied and characterized are trypsin, plasmin and the organomercurials. These agents appear to have in common an ability to disrupt the structure of the stable latent enzyme in such a way as to allow the generation of a proteolytic active site. In the case of organomercurial activation, intramolecular proteolytic cleavage of the amino-terminus of the enzyme occurs subsequent to generation of activity. A similar intramolecular process is seen with trypsin and plasmin activation except that it is initiated by a single trypsin or plasmin catalyzed cleavage in the amino-terminus prior to the autocatalytic cleavages. A possible explanation for organomercurial activation is that the mercurial disrupts a cysteinyl residue coordination bond with the active site zinc that prevents interaction with substrate. Disruption of this complex would allow productive enzyme-substrate interaction via the newly available coordination site. In addition, activated stromelysin is capable of increasing the specific activity of active interstitial collagenase by approximately ten-fold through what appears to be proteolytic removal of a small peptide.
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PMID:Activation of extracellular matrix metalloproteases by proteases and organomercurials. 148 30

The purification and cloning of a novel metalloproteinase inhibitor (MI or TIMP-2) related to tissue inhibitor of metalloproteinases (TIMP) has been recently described by our laboratory (DeClerck, Y.A., Yean, T. D., Ratzkin, B.J., Lu, H.S., and Langley, K.E. (1989) J. Biol. Chem. 264, 17445-17453; Boone, T.C., Johnson, M.J., DeClerck, Y.A., and Langley, K.E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2800-2804). We have transfected Chinese hamster ovary cells with a vector containing human MI/TIMP-2 cDNA and purified recombinant-derived MI/TIMP-2 (rMI/rTIMP-2) from the conditioned medium of such cells. We have investigated the inhibitory activity of rMI/rTIMP-2 toward rabbit fibroblast interstitial collagenase. The inhibition of activated collagenase by rMI/rTIMP-2 is stoichiometric and consistent with the formation of a 1:1 molar ratio complex. In addition to blocking the activated enzyme, rMI/rTIMP-2 inhibits the conversion of 52-kDa procollagenase to the 42-kDa active enzyme initiated by organomercurials. When plasmin is used as activator, rMI/rTIMP-2 does not inhibit the plasmin-mediated conversion of the 52-kDa proenzyme to the 46-kDa inactive intermediate but blocks further conversion of the 46-kDa intermediate to the 42-kDa active enzyme. The data indicate that rMI/rTIMP-2 blocks the autoproteolytic activation of procollagenase. Also, rMI/rTIMP-2 forms complexes with the 52-kDa procollagenase, the 46-kDa intermediate, and with the 42-kDa activated enzyme which are stable to sodium dodecyl sulfate (SDS), such that the complexes can be visualized by SDS-polyacrylamide gel electrophoresis. It appears that the formation of a SDS-stable complex with procollagenase requires an initial conformational change of the procollagenase brought about by organomercurials or by plasmin cleavage. The data suggest that MI/TIMP-2 may be able to control the extracellular action of certain metalloproteinases not only at the level of the activated enzyme but also at the level of proenzyme activation.
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PMID:Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. 184 92

Implantation and subsequent placental development in many species including the human are dependent on trophoblast invasion of the uterine epithelium, the underlying basement membrane, connective tissue and blood vessels. However, trophoblast invasion in situ is strictly controlled by the microenvironment provided by the pregnant uterus. Key mechanisms underlying various steps in trophoblast invasion of basement membrane and stroma are similar to those identified in the case of invasive tumor cells: (a) attachment to basement membrane by binding to laminin and possibly other basement membrane components; (b) detachment from the basement membrane matrix prior to its penetration, a process that requires the presence of complex-type oligosaccharides on the cell surface; (c) breakdown of basement membrane components by trophoblast-derived metalloproteases (type IV and interstitial collagenase) and serine proteases (plasminogen activator). Type IV collagenase activity is stimulated by binding to laminin, a molecule also secreted by the trophoblast. Activation of trophoblast-derived metalloproteases appears to be plasmin-dependent. Plasmin results from the cleavage of plasminogen by trophoblast-derived plasminogen activator. Control of trophoblast invasion in situ is mediated by decidua-derived transforming growth factor beta (TGF beta) which in turn induces tissue inhibitor of metalloproteases (TIMP) both in the decidua and the trophoblast. We suggest that this control of trophoblast invasiveness is regulated both spatially as well as temporally during gestation. A preprogrammed decline in trophoblast invasiveness with increasing gestational age remains an additional possibility. The nature of the loss of control of trophoblast invasiveness in choriocarcinoma remains to be identified. Refractoriness to TGF beta action remains to strong possibility.
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PMID:Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. 209 85

After specific chemotherapy, granulomatous fibrosis undergoes a marked reversal in liver of Schistosoma mansoni-infected mice. We have previously shown that this fibrosis reversal was related to a high proportion of the active form of the interstitial collagenase. In vitro, plasmin has been described as a physiological activator of interstitial procollagenase. Moreover, plasmin itself degrades directly matrix components such as proteoglycans and fibronectin. We have thus followed the course of the plasminogen activator, which converts plasminogen zymogen to plasmin, in liver of S. mansoni-infected mice treated with praziquantel, as schistosomicidal drug. It was found that plasminogen activator activity in the liver increases rapidly until 5 days after treatment as compared to nontreated infected mice and then diminishes gradually. Increased plasminogen activator activity appears to be one of initial events leading to this fibrosis reversal.
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PMID:Plasminogen activator activity increases during reversal of hepatic fibrosis in murine schistosomiasis. 211 35

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.
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PMID:In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. 246 98

We present a cascade of proteolytic events catalyzed by the proteases secreted by cultured keratinocytes and fibroblasts that results in the activation of interstitial procollagenase. Cultured human skin fibroblasts constitutively secrete interstitial collagenase and stromelysin as proenzymes. In contrast, interstitial collagenase found in serum-free skin organ culture conditioned medium is activated. Cocultivation of the major cellular components of skin organ culture, dermal fibroblasts and epidermal keratinocytes, induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a urokinase-dependent pathway where added keratinocytes secrete the plasminogen activator urokinase, which converts plasminogen into plasmin. Plasmin is capable of activating purified procollagenase and prostromelysin. Plasmin-dependent activation of procollagenase generates an enzyme species, by amino-terminal processing, identical to those generated by limited proteolysis with trypsin or treatment with organomercurial compounds. Catalytic amounts of activated stromelysin can in turn convert plasmin- or trypsin-activated collagenase into a fully active enzyme by removal of approximately 15 amino acid residues from the carboxyl end of the enzyme. This results in a 5- to 8-fold increase in collagenase specific activity that is due to its proteolytic cleavage and not to the presence of the activator stromelysin. Stromelysin alone in both pro- and activated forms is not capable of efficient activation of human fibroblast interstitial procollagenase.
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PMID:Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. 246 56

Tumor growth is dependent upon angiogenesis. There is an intense search for pharmacological inhibitors of angiogenesis as a novel approach to treat angiogenic diseases, e.g., arthritis, diabetic retinopathy or cancer. A series of compounds, originally studied as potential protein kinase C inhibitors, included the diaminoanthraquinone NSC 639366 (1-[[3-(diethylamino)-2-hydroxypropyl]amino]-4-[(2,3- epoxypropyl)amino]-9,10-anthracenedione fumaric acid salt) (SPC-100097), was found to reversibly inhibit bovine endothelial cell growth with an IC50 that ranged between 1 and 4 nM. NSC 639366 reversibly inhibited endothelial cell migration, particularly endothelial cells stimulated by the potent angiogenic molecule, basic fibroblast growth factor. The activity of secreted urokinase-type plasminogen activator and active interstitial collagenase, but not gelatinase, was inhibited by NSC 639366. In vivo, angiogenesis was significantly inhibited by NSC 639366 by using the chick chorioallantoic membrane or the rat corneal bioassay. Two analogs of NSC 639366 did not inhibit endothelial cell growth. These experiments introduce a novel compound that could be clinically useful against angiogenic diseases and encourage further development of compounds that inhibit the plasminogen-plasmin system known to be a key regulator of angiogenesis.
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PMID:A diaminoantraquinone inhibitor of angiogenesis. 752 34


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