Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cathepsin L proteinase secreted by the parasitic helminth Fasciola hepatica can cleave fibrinogen and produce a fibrin clot with a specific activity of 4.7 National Institutes of Health thrombin-equivalent U/mg. This is the first report of a fibrinogen-clotting activity aside that of thrombin and the snake venom proteinases, which are all serine proteinases. Clot formation by cathepsin L is not inhibited by the thrombin inhibitor hirudin or by the anti-polymerant H-Gly-Pro-Arg-Pro-OH. The enzyme exerts its activity on fibrinogen in a unique manner. Although the cleavage of fibrinogen may involve the initial removal of fibrinopeptides, additional proteolysis of the alpha, beta and gamma fibrinogen polypeptides takes place. SDS/PAGE analysis of the cathepsin-L-produced clots revealed that cleavage of the alpha polypeptide (66 kDa) precedes that of the beta (52 kDa) and gamma (46.5 kDa) polypeptides. Concurrent with the cleavage of these polypeptides is the appearance of components of 120, 100 and 25 kDa. The appearance of higher molecular-sized components in the cathepsin L clots suggests that polymerisation involves the formation of molecular interactions that are resistant to boiling in mercaptoethanol and SDS.
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PMID:Fasciola hepatica cathepsin L proteinase cleaves fibrinogen and produces a novel type of fibrin clot. 755 57

Human lung macrophages express all four of the known lysosomal thiol proteases: cathepsins B, H, L, and S. These enzymes share a similar size and targeting mechanism for lysosomal accumulation and all have relatively indiscriminate substrate specificity in comparison with such highly selective serine proteases as urokinase or thrombin. These enzymes do have distinctive properties: only cathepsin B has C-terminal dipeptidase activity, only cathepsin H has potent aminopeptidase activity, and only cathepsin L and S are elastolytic. Cathepsin S is unique in that it is stable at neutral pH; indeed, at neutral pH it has elastolytic activity roughly comparable with that of neutrophil elastase. Recent studies of the differential expression of these cathepsins suggest they not only cooperate in terminal degradation of endocytized protein but also have specific functions such as proenzyme activation, antigen processing, and tissue remodeling, especially bone matrix resorption. Lysates of lung macrophages degrade elastin at neutral pH, suggesting that necrosis of macrophages at sites of macrophage accumulation, e.g., caseation necrosis, could contribute to tissue destruction. Tissue destruction and remodeling by thiol proteases expressed by live macrophages, however, is limited by tight compartmentalization of cathepsins to lysosomes. Nonetheless, macrophages accumulate at sites of known injury in cigarette smokers. Because these cells contain potent elastases, and because lysosomal enzyme release and cell surface acidification are regulated events, dysregulation of thiol protease expression in stimulated macrophages may contribute to the injury observed in cigarette smokers with non-alpha-1-protease inhibitor-type emphysema.
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PMID:The role of thiol proteases in tissue injury and remodeling. 795 52

By focusing on the amphiphilic properties of cyclopropenone (e.g. a good electrophile and a precursor for a stable 2pi-aromatic hydroxycyclopropenium cation), a new class of cysteine proteinase inhibitors containing a cyclopropenone moiety was designed. For the purpose of the present research, we needed to devise a new method to introduce a peptide-related moiety as a substituent on the cyclopropenone residue. We investigated the reaction of metalated cyclopropenone acetal derivatives (2, R2 = metal) with N-protected alpha-aminoaldehydes 4 to obtain the adduct 5, and succeeded in the preparation of highly potentiated cysteine proteinase inhibitors 8 after several steps transformations. They showed strong inhibitory activities only to cysteine proteinases such as calpain, papain, cathepsin B, and cathepsin L and not to serine (e.g. thrombin and cathepsin G) and aspartic proteinases (e.g. cathepsin D). Kinetic studies indicated that they are competitive inhibitors, and by the examinations of their inhibitory mechanism it became clear that they are reversible inhibitors.
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PMID:Cyclopropenone-containing cysteine proteinase inhibitors. Synthesis and enzyme inhibitory activities. 1035 36

In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.
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PMID:Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli. 1039 23

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
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PMID:Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. 1101 86

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
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PMID:Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. 1137 81

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.
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PMID:Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies. 1600 43

The Western corn rootworm is one of the most economically important pests in corn. One possibility for controlling this pest is the cultivation of transgenic corn expressing Bacillus thuringiensis (Bt) toxins, such as Cry3A, Cry34Ab1/Cry35Ab1, and Cry3Bb1. However, widespread cultivation of the resulting Bt corn may result in the development of resistant pest populations. The Bt toxins are processed by proteases in the midgut of susceptible insects. Thus, protease activity studies were conducted using the midgut juice (pH 5.75) from third instars larvae of the susceptible Western corn rootworm. As a result, the activities of the serine endopeptidases trypsin, chymotrypsin, elastase, cathepsin G, plasmin, and thrombin; the cysteine endopeptidases cathepsin L, papain, cathepsin B, and cathepsin H; the aspartic endopeptidase pepsin; the metallo endopeptidase saccharolysin; the exopeptidase aminopeptidase, and the omegapeptidase acylaminoacylpeptidase were detected. These results are of basic interest but also lead to reference systems for the identification of protease-mediated resistance mechanisms in potentially resistant individuals.
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PMID:Protease activities in the midgut of Western corn rootworm (Diabrotica virgifera virgifera LeConte). 1932 44

The glycosaminoglycan heparin is known to possess antimetastatic activity in experimental models and preclinical studies, but there is still uncertainty over its mechanism of action in this respect. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III, but a similar cofactor role has not been previously investigated for proteases linked to metastasis. The squamous cell carcinoma antigens (serpins B3 and B4) are tumor-associated proteins that can inhibit papain-like cysteine proteases, including cathepsins L, K, and S. In this study, we show that SCCA-1 (B3) and SCCA-2 (B4) can bind heparin as demonstrated by affinity chromatography, native PAGE gel shifts, and intrinsic fluorescence quenching. Binding was specific for heparin and heparan sulfate but not other glycosaminoglycans. The presence of heparin accelerated inhibition of cathepsin L by both serpins, and in the case of SCCA-1, heparin increased the second order inhibition rate constant from 5.4 x 10(5) to >10(8), indicating a rate enhancement of at least 180-fold. A templating mechanism was shown, consistent with ternary complex formation. Furthermore, SCCA-1 inhibition of cathepsin L-like proteolytic activity secreted from breast and melanoma cancer cell lines was significantly enhanced by heparin. This is the first example of glycosaminoglycan enhancement of B-clade serpin activity and the first report of heparin acting as a cofactor in serpin cross-class inhibition of cysteine proteases. Most importantly, this finding raises the possibility that the anticancer properties of heparin may be due, at least partly, to enhanced inhibition of prometastatic proteases.
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PMID:Heparin enhances serpin inhibition of the cysteine protease cathepsin L. 1995 74

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K(i)=3.29 nM) and human cathepsin L (K(i)=3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.
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PMID:Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi. 2187 78


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