Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.
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PMID:Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins. 846 59

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC.
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PMID:Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. 954 62

Interleukin-2 (IL-2) is a potent activator of cellular immunity and has been utilized as an immunotherapeutic agent. We stably immobilized human IL-2 to collagen by covalently binding it to the N-terminus of human type III collagen (3A1) as IL2-3A1 chimeric protein using recombinant technology. The present study was aimed at liberating IL-2 from the immobilized chimeric protein by treating the chimera with bacterial collagenase. These IL2-3A1 chimeras were synthesized in insect cells which had been infected with baculovirus vectors carrying IL2-3A1 cDNA. The IL2-3A1 protein produced was shown to be in a pepsin-resistant triple helical structure and exhibited IL-2 activity to a similar extent as IL-2 itself. IL2-3A1 could be immobilized on the surface of plastic dishes by incubating it in the dishes. The IL-2 region of the immobilized IL2-3A1 was liberated to culture media by collagenase treatment and this freed IL-2 stimulated the growth of lined T cells. Thus, IL2-3A1 chimeric protein could be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment.
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PMID:Interleukin-2-collagen chimeric protein which liberates interleukin-2 upon collagenolysis. 1203 63

Ewing sarcoma is a malignant bone and soft tissue tumor of children and young adults, which is known to be highly aggressive and invasive. It expresses specific chimeric genes (EWS-FLI-1, EWS-ERG, EWS-ETV1, and EWS-E1AF), the 3' portions of which are all members of the ETS family. ETS-related proteins, such as FLI-1, ERG, and E1AF, transactivate the promoters of matrix metalloproteinase (MMP) genes, which play important roles in the processes of invasion and metastasis. Therefore, we hypothesize that the Ewing sarcoma-specific chimeric genes also transactivate the MMP genes, contributing to the tumor's invasiveness and propensity for metastasis. To verify this hypothesis, we investigated the expression of MMPs in eight Ewing sarcoma cell lines. Surprisingly, MMP-1 and MMP-3 were not expressed at all in any of the cell lines. MMP-9 was expressed in four out of the eight cell lines, and MMP-2 and MT1-MMP in all of the cell lines. Ewing sarcoma-specific chimeric genes have been shown to transactivate the promoter of the MMP-1 gene by the reporter assay, and bind to the putative recognition sites in the MMP regulatory elements by the gel shift assay. However, an in vivo formaldehyde cross-linking study revealed that the chimeric protein did not bind to the predicted ETS recognition sites in the regulatory elements of the MMPs. These results indicate that the absence of the MMP expression in the tumor cells is at least in part due to the loss of accessibility of the ETS recognition sites in the regulatory elements of the MMP genes. Therefore, we should be careful before theorizing simply that a putative binding site is essential for the transcription of critical genes, since the binding of this fusion protein was found to be modulated in tumor cells in this study.
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PMID:Lack of matrix metalloproteinase (MMP)-1 and -3 expression in Ewing sarcoma may be due to loss of accessibility of the MMP regulatory element to the specific fusion protein in vivo. 1205 64

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. We now examine a more limited amino-terminal region of alpha3(IV) non-collagenous domain (NC1) and the impact of single amino acid (AA) mutations of this region on glomerulonephritis induction. Rats were immunized with collagenase-solubilized glomerular basement membrane (csGBM), D3, an alpha1(IV)NC1 chimeric protein with 69 AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (non-binding), P9, P10, single AA mutants (non-binding), and S2, alpha1(IV)NC1 with 9 AA of alpha3(IV)NC1 (binding). All rats immunized with csGBM and S2 and 50% of D3 rats developed glomerulonephritis. csGBM rats had intense GBM-bound IgG deposits, but S2 and D3 rats had minimal deposits. None of the D4, P9, or P10 rats developed glomerulonephritis. Lymphocytes from nephritic rats proliferated with csGBM, S2, and D3, but not with D4, P9, or P10. Discrete segments of alpha3(IV)NC1 within the alpha1(IV)NC1 backbone can induce glomerulonephritis. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis. These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. Further, they define a critical limited AA sequence within alpha3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic alpha1(IV)NC1 without in vivo antibody binding. This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man.
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PMID:Point mutations of single amino acids abolish ability of alpha3 NC1 domain to elicit experimental autoimmune glomerulonephritis in rats. 1297 Mar 56

Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.
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PMID:Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system. 2437 78

The matrix metalloproteinases (MMP) are endopeptidases performing proteolytic functions in the extracellular matrix and their overexpression has been suggested to be a characteristic of malignant tumors. Molecular changes such as the presence of chimeric protein Ewing's sarcoma protein-friend leukemia virus integration 1 (EWS-FLI1) in the Ewing family of tumors (EFT) and the oncogenes C-ERBB-2, N-MYC, C-MYC in medulloblastoma (MB) promote the overexpression of MMP. In the present study, protein expression of MMP-1, -2, -3, -9 and -14 was qualitatively evaluated in 17 EFT and MB samples of children and adolescent by western blotting and optical densitometry, and the level of gene expression of some MMPs was determined by real-time quantitative polymerase chain reaction. Five MB samples (45.4%) presented expression of the five MMPs and six samples (54.6%) presented expression of at least one of them. Four EFT samples (66.6%) presented expression of MMP-2, -9 and -14, and two samples (33.4%) presented expression of at least one of these MMPs, whereas the presence of MMP-1 and -3 was not observed. Gene analysis showed that MMP-2 had a high expression in MB, while the expression of MMP-9 and MMP-14 was higher in EFT. It has been established that the expression of the MMPs might be related to a complex pathway of gene regulation.
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PMID:Protein expression of matrix metalloproteinase (MMP-1, -2, -3, -9 and -14) in Ewing family tumors and medulloblastomas of pediatric patients. 2762 20