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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extracellular matrix metalloproteases (MMPs) secreted by various human tumor cells play a crucial role in tumor cell invasion and metastasis, but their expression in malignant mesothelioma (MM) cells has not been examined. In this study, we have investigated the spectrum of MMPs and tissue inhibitors of metalloproteases (TIMPs) produced by 8 MM cell lines. Using RT-PCR, we found that all investigated MM cell lines expressed genes encoding mRNA for MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and TIMPs 1, 2 and 3. We also found that 6/8 MM cell lines expressed MMP-7 (matrilysin) and 3/8 MM cell lines expressed MMP-10 (stromelysin-2). MMP-11 (stromelysin-3) was not detected in any of the MM cell lines. Production of MMP-2 and MMP-9 was confirmed using gelatin zymography. In addition, all MM cell lines secreted a 66 kDa metalloprotease, while 3/8 MM cell lines secreted 46, 48, 51 and 63 kDa metalloproteases which specifically degraded the extracellular matrix components fibronectin, vitronectin and laminin. The 66 kDa protease was identified as MMP-3 by Western blot. Our results reveal a broad spectrum of MMPs and TIMPs produced by MM cells and indicate that different substrate specificities of MMPs may play a role in MM cell invasion.
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PMID:Expression and activity of matrix metalloproteases in human malignant mesothelioma cell lines. 1126 73

The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.
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PMID:Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04. 1186 35

Intracellular signals generated by mechanical strain profoundly affect the metabolic function of osteoblast-like periodontal ligament (PDL) cells, which reside between the tooth and alveolar bone. In response to applied mechanical forces, PDL cells synthesize bone-resorptive cytokines to induce bone resorption at sites exposed to compressive forces and deposit bone at sites exposed to tensile forces in an environment primed for catabolic processes. The intracellular mechanisms that regulate this bone remodeling remain unclear. Here, in an in vitro model system, we show that tensile strain is a critical determinant of PDL-cell metabolic functions. Equibiaxial tensile strain (TENS), when applied at low magnitudes, acts as a potent antagonist of interleukin (IL)-1beta actions and suppresses transcriptional regulation of multiple proinflammatory genes. This is evidenced by the fact that TENS at low magnitude: (i) inhibits recombinant human (rh)IL-1beta-dependent induction of cyclooxygenase-2 (COX-2) mRNA expression and production of prostaglandin estradiol (PGE2); (ii) inhibits rhIL-1beta-dependent induction matrix metalloproteinase-1 (MMP-1) and MMP-3 synthesis by suppressing their mRNA expression; (iii) abrogates rhIL-1beta-induced suppression of tissue inhibitor of metalloprotease-II (TIMP-II) expression; and (iv) reverses IL-1beta-dependent suppression of osteocalcin and alkaline phosphatase synthesis. Nevertheless, these actions of TENS were observed only in the presence of IL-1beta, as TENS alone failed to affect any of the aforementioned responses. The present findings are the first to show that intracellular signals generated by low-magnitude mechanical strain interfere with one or more critical step(s) in the signal transduction cascade of rhIL-1beta upstream of mRNA expression, while concurrently promoting the expression of osteogenic proteins in PDL cells.
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PMID:Signaling by mechanical strain involves transcriptional regulation of proinflammatory genes in human periodontal ligament cells in vitro. 1193 44

Many virulence factors are secreted by the gram-positive, spore forming bacterium Bacillus cereus. Most of them are regulated by the transcriptional activator, PlcR, which is maximally expressed at the beginning of the stationary phase. We used a proteomic approach to study the impact of the PlcR regulon on the secreted proteins of B. cereus, by comparing the extracellular proteomes of strains ATCC 14579 and ATCC 14579 Delta plcR, in which plcR has been disrupted. Our study indicated that, quantitatively, most of the proteins secreted at the onset of the stationary phase are putative virulence factors, all of which are regulated, directly or indirectly, by PlcR. The inactivation of plcR abolished the secretion of some of these virulence factors, and strongly decreased that of others. The genes encoding proteins that are not secreted in the DeltaplcR mutant possessed a regulatory sequence, the PlcR box, upstream from their coding sequence. These proteins include collagenase, phospholipases, haemolysins, proteases and enterotoxins. Proteins for which the secretion was strongly decreased, but not abolished, in the DeltaplcR mutant did not display the PlcR box upstream from their genes. These proteins include flagellins and InhA2. InhA2 is a homologue of InhA, a Bacillus thuringiensis metalloprotease that specifically degrades antibacterial peptides. The mechanism by which PlcR affects the production of flagellins and InhA2 is not known.
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PMID:Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. 1211 62

The crystal structure of a collagen-binding domain (CBD) with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 A resolution in the absence of calcium (1NQJ) and at 1.65 A resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the CBD reveals a beta-sheet sandwich fold with the linker adopting an alpha-helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the collagen-binding surface of the protein and propose likely collagen-protein binding models.
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PMID:A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. 1268 7

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.
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PMID:Expression and function of the Trypanosoma brucei major surface protease (GP63) genes. 1270 78

African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658-24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.
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PMID:Surface coat remodeling during differentiation of Trypanosoma brucei. 1271 4

Marimastat [BB 2516, TA 2516] is a second-generation anticancer drug originally developed with British Biotech in Europe and North America. It is an orally active metalloprotease inhibitor of the same class as batimastat, and is the first compound in this class to have completed a pivotal clinical trial. Marimastat also has collagenase- and angiogenesis-inhibiting properties. British Biotech and Schering-Plough have signed an agreement enabling the latter to develop and market marimastat in North America and Europe. Under the terms of the agreement, British Biotech will receive an up-front license fee of 4 million US dollars and a 4 million US dollars equity investment in British Biotech by Schering-Plough. Schering-Plough holds rights to marimastat in all countries other than the Far East and Japan. The two companies are considering asking the FDA for accelerated approval in gastric cancer based on the secondary endpoint of progression-free survival. Marimastat is licensed to Tanabe Seiyaku in Japan, where phase II clinical trials are underway for the treatment of advanced gastric cancer and lung cancer. Further phase II trials in other tumour types are planned. The commencement of phase II trials in Japan resulted in a milestone payment of 5 million US dollars to British Biotech from Tanabe Seiyaku. Tanabe Seiyaku also holds rights to marimastat in the Far East. Marimastat has been in pivotal phase III trials in glioblastoma, breast, ovarian and small and non-small cell lung cancer, but these trials have all been discontinued because marimastat failed to show superior efficacy over either standard chemotherapy or placebo. Results from the marimastat 131 trial in patients with glioblastoma, for example, indicated that marimastat was no better than placebo at prolonging survival in these cancer patients. In June 2000, when the results of this study were released, shares in British Biotech fell 21.6% to just 19 pence per share. The phase III trial in small cell lung cancer was discontinued when the results of study 140 were released in February 2001 showing that marimastat was not significantly more effective than placebo in prolonging the survival of small cell lung cancer patients. The results of this study were consistent with those reported in study 117. British Biotech has also conducted a phase III placebo-controlled study of marimastat as monotherapy in patients with inoperable gastric cancer at 37 centres throughout Europe. Results from this trial indicated that it did not achieve its primary endpoint of a statistically significant survival benefit over placebo. However, data collected during the follow-up period have shown increases in survival benefit in the treatment group in addition to a significant improvement in disease-free progression, the secondary endpoint of the trial. Development of marimastat for this indication is ongoing. In May 2001, British Biotech reported data from an interim analysis of results from the remaining phase III study in pancreatic cancer (study 183) that showed no patient benefit for marimastat recipients compared with gemcitabine. However, these results did not meet stopping criteria and the study continues under the guidance of Schering-Plough. The multicentre trials are being conducted in the US, Canada and the European Union. The phase III trial of marimastat in combination with carboplatin that was being conducted in patients with ovarian cancer was discontinued because British Biotech realised that the design of the trial was insufficient for registration in the US or Europe. Altogether, seven phase III studies have failed to meet their primary end-points, but the company has stated that the effectiveness of marimastat is more likely to be seen in patients with less advanced disease. Phase II trials in prostate and head and neck cancer are still underway in the US.
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PMID:Marimastat: BB 2516, TA 2516. 1275 9

Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.
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PMID:Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly. 1276 Nov 82

To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37 degrees C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3' end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57-42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.
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PMID:Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C-terminal deletions. 1282 1


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