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 actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.
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PMID:Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. 821 28

Human growth hormone (hGH) inhibits alpha 1(I) collagen gene expression in cultured avian skin fibroblasts resulting in a decrease in the amount of collagenase-digestible proteins (CDP) in the medium. In addition, a synergism exists between GH and insulin-like growth factor-I (IGF-I) in their effect on CDP. Four N-terminal modified hGH analogs were tested for their ability to affect collagen metabolism in these cells. The truncated analog Des-7 hGH(R8M, D11A) was found to be a strong antagonist of the hGH-induced inhibition of the collagen synthesis but by itself did not inhibit collagen alpha 1(I) gene expression or modify the CDP appearance in the medium. Some synergism between Des-7 hGH and IGF-I was observed. The analog Met-hGH(R19H, L20P), in which Arg19 was replaced by histidine, and Leu20 by proline was only partially potent compared with the native hormone in causing inhibition of collagen gene expression, in attenuating CDP appearance in the medium, and in antagonizing hGH. However, this analog was as potent as hGH in its ability to synergize with IGF-I. The importance of His18 was assessed by testing the response to Met-hGH(H18D), in which His18 was replaced by Asp, and to Met-hGH(H18Q), in which His18 was replaced by glutamine (as in chicken GH sequence). Substitution of His18 by a negatively charged amino acid abolished all the hormone activities tested whereas substitution with glutamine restored only part of the activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of N-terminal modified analogs of growth hormone on collagen synthesis in avian skin fibroblasts. 831 27

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U2OS cells. The collagenous nature of this matrix was demonstrated by the incorporation of [3H]proline and its subsequent release by purified collagenase. Both U2OS matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U2OS matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against alpha 2 or beta 1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn2+ and Mg2+ ions but not by Ca2+ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor beta (TGF-beta), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of alpha 2 beta 1 integrin on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-beta suggests that the co-expression of TGF-beta and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.
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PMID:Bone cell matrix promotes the adhesion of human prostatic carcinoma cells via the alpha 2 beta 1 integrin. 852 12

Tissue inhibitor of metalloproteinases (TIMPs) are secreted proteins that regulate the activity of metalloproteinases, enzymes important in development, tissue remodeling, angiogenesis, and tumorigenesis. To assess the importance of three highly conserved amino acids, His7, Asp16, and His95, in determining the biological properties of mouse TIMP-1, they were mutated into Arg, Tyr, and Arg, respectively. Recombinant vectors constructed to express the wild-type and mutant TIMP-1 proteins under the control of the metallothionein promoter were transfected into mouse melanoma B16F10 cells, which produce very little TIMP-1. Individual clones were isolated and characterized by Southern, Northern, and Western blotting to verify the presence of the TIMP-1 minigene and its expression. Analyses of conditioned media for collagenase-inhibiting activity indicated that both histidine mutants, but not the aspartic acid mutant, were functionally impaired. An investigation of the cell migration, matrix invasion, and tumor formation capabilities of several individual clones representing each of the mutants revealed that the His7Arg and His95Arg mutations, but not the Asp16Tyr mutation, largely abolished the ability of the protein to inhibit all of these activities. These data establish that for B16F10 cells, endogenously generated TIMP-1 is an effective inhibitor not only of matrix invasion and tumorigenicity but also, unexpectedly, of cell motility on plastic. The novel finding that both His7 and His95 are separately essential for significant TIMP-1 activity in vivo provides an important new insight into TIMP-1 function.
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PMID:Directed mutagenesis reveals that two histidines in tissue inhibitor of metalloproteinase-1 are each essential for the suppression of cell migration, invasion, and tumorigenicity. 893 Apr 8

The initial interaction of the human osteoblast-like cell line Saos-2 with orthopaedic implant materials was analyzed to determine the mechanism by which these cells adhere to implant surfaces. Saos-2 cells were allowed to attach to disks composed of the orthopaedic implant materials Tivanium (Ti6A14V) and Zimaloy (CoCrMo) and to control disks of glass and plastic. Serum had no effect on the number of cells that attached to Tivanium and Zimaloy at 4 or 24 hours but did increase the number of cells that attached to glass at 24 hours. Collagen synthesis was determined by [3H]proline incorporation into collagenase-digestible protein and noncollagen protein. A significant increase of 19% was found for collagen synthesized in cells cultured on Zimaloy for 24 hours compared with glass, with no differences on Tivanium and plastic. However, collagenase-digestible protein and noncollagen protein were increased the most (204 and 198%, respectively) on Tivanium compared with glass. To determine if integrins were involved in cell attachment to implant materials, the peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), which blocks integrin receptors through the Arg-Gly-Asp sequence, was added to the cells in serum-free medium. This peptide inhibited cell adhesion by 28% on Tivanium and 40% on Zimaloy but had no effect on glass and plastic. The control peptide GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) had no effect on adhesion. Inhibition of protein synthesis and enzymatic removal of surface proteins did not affect the ability of Arg-Gly-Asp peptides to inhibit cell attachment to the implant materials. These results suggest that integrins are able to bind directly to Tivanium and Zimaloy. Western blot analysis of integrin protein demonstrated changes in many integrin subunits, depending on the substrate to which cells attached. In particular, the beta 1 integrin subunit was increased 3.8 to 9.5-fold at 24 hours. To determine specifically which integrins may be involved in adhesion, antibodies to integrins were added. An antibody to the fibronectin receptor, alpha 5 beta 1, significantly inhibited binding of cells to Tivanium by 63% and to Zimaloy by 49% and had no effect on glass. The vitronectin receptor antibody, alpha v beta 3/beta 5, did not alter cell adhesion. In conclusion, osteoblast-like cells appear to be capable of attaching directly to implant materials through integrins. The type of substrate determines which integrins and extracellular matrix proteins are expressed by osteoblasts. These data provide information on how implant materials may affect osteoblast differentiation and bone growth.
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PMID:Response of human osteoblasts to implant materials: integrin-mediated adhesion. 898 29

The heterogeneity of urinary degradation products of C-terminal telopeptides derived from the alpha1 chain of human type I collagen was investigated and characterized. The urinary fragments characterized in this study consisted of two cross-linked (X) amino acid sequences derived from the C-terminal telopeptide (alpha1) of type I collagen. Fragments containing the sequence EXAHDGGR, with a DG site being either nonisomerized (Asp-Gly) or beta-isomerized (betaAsp-Gly), were identified. Pyridinoline was detected among the pyridinium cross-links, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that of galactosyl pyridinoline. A nonfluorescent cross-link was also found. The concentration of fragments derived from the C-terminal telopeptide region of type I collagen containing the sequence Asp-Gly (alphaCTX) and/or betaAsp-Gly (betaCTX) was measured by enzyme-linked immunosorbent assays in urine and in collagenase digests of trabecular and cortical bone of young and old origin. It was shown that the urinary ratio between such fragments, alphaCTX/betaCTX, was higher in children compared with adults and that the ratio decreased with increasing age of bone. The results indicated that the C-terminal telopeptide fragments derived from type I collagen excreted into urine originated mainly from bone. In conclusion, it is demonstrated for the first time that the C-terminal telopeptide alpha1 chain of type I collagen contains an Asp-Gly site prone to undergo beta-isomerization and that the degree of beta-isomerization of this linkage apparently increases with increasing age of bone. These findings indicate that the ratio alphaCTX/betaCTX might be clinically important in diagnosing metabolic bone diseases.
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PMID:Characterization of urinary degradation products derived from type I collagen. Identification of a beta-isomerized Asp-Gly sequence within the C-terminal telopeptide (alpha1) region. 909 8

Decorin (DCN) is a ubiquitous proteoglycan comprised of a core protein attached to a single dermatan/chondroitin sulphate glycosaminoglycan chain. It may play a role in regulation of collagen fibrillogenesis and function as a reservoir of transforming growth factor beta (TGF-beta) in the extracellular milieu. We have examined the susceptibility of DCN to five different matrix metalloproteinases (MMPs): MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin) and MMP-9 (gelatinase B). MMP-2 and MMP-3 digest DCN into seven major fragments in a similar pattern. The N-terminal sequence of the two fragments generated by MMP-2 and MMP-3 is Leu211-Lys-Gly-Leu-Asn, but that of the others is Asp1-Glu-Ala-Ser-Gly. MMP-7 cleaves DCN into three major fragments which have the N-termini Asp1-Glu-Ala-Ser-Gly, Glu2-Ala-Ser-Gly-Ile and Leu244-His-Leu-Asp-Asn. Activities of MMP-1 and MMP-9 against DCN are negligible. The values of Km for the MMPs capable of degrading DCN are very similar (10-12 microM), but the kcat/Km value for MMP-7 (30.5 microM-1.h-1) is 4.5-fold higher than those for MMP-2 and MMP-3. Incubation of a DCN-TGF-beta1 complex with MMP-2, -3 or -7 results in release of TGF-beta1 from the complex. These data indicate proteolytic degradation of DCN by MMP-2, MMP-3 and MMP-7, and suggest the possibility that, under pathophysiological conditions, the digestion by the MMPs may induce tissue reactions mediated by TGF-beta1 released from DCN in the connective tissues.
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PMID:Degradation of decorin by matrix metalloproteinases: identification of the cleavage sites, kinetic analyses and transforming growth factor-beta1 release. 914 53

Osteoclast-like multinucleated cells (OCLs) were prepared on collagen gels in a coculture system of mouse bone marrow cells and osteoblasts, and purified by collagenase and a subsequent pronase treatment. More than 80% of the purified OCLs were found to undergo apoptotic cell death by 48 h during the culture in a culture medium containing 10% fetal bovine serum (FBS). Withdrawal of FBS from the culture medium accelerated the cell death, which induced more than 80% of OCLs to undergo apoptotic cell death by as early as 18 h. Two peptide inhibitors of caspases (interleukin-1beta-converting enzyme family proteases), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), extended the survival time of OCLs in the presence and absence of 10% FBS, but the effect was rather limited in the absence of FBS. Because interleukin-1alpha (IL-1alpha) and the macrophage colony stimulating factor (M-CSF) are known to promote the survival of osteoclasts, we examined the effect of the peptide inhibitors and these cytokines. Combinations of the peptide inhibitors and IL-1alpha, or the peptide inhibitors and M-CSF, were more effective than the inhibitors alone. When endogenous caspase activities of OCLs were analyzed using fluorescence peptide substrates, the activities, in particular, caspase-3 (CPP32)-like activity, were markedly increased in OCLs by the withdrawal of FBS from the culture medium. IL-1alpha and M-CSF suppressed the activation of the caspases. In addition, western blot analysis revealed that the expression of Bcl-2, which inhibits the activation of caspases, was very weak or even negligible in OCLs. Taken together, these results suggest that the caspases are involved in the regulation of survival and apoptotic cell death of osteoclasts.
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PMID:Caspases (interleukin-1beta-converting enzyme family proteases) are involved in the regulation of the survival of osteoclasts. 966 28

A series of tetrapeptide p-nitroanilide substrates of the general formula: suc-Ala-Ala-Pro-Aaa-p-nitroanilide was used to map the S1 binding pocket of human cathepsin G. Based on the kcat/Km parameter, the following order of preference was found: Lys=Phe>Arg=Leu>Met>Nle=Nva>Ala>Asp. Thus, the enzyme exhibits clear dual and equal trypsin- and chymotrypsin-like specificities. Particularly deleterious were beta-branched side chains of Ile and Val. The P1 substrate preferences found for cathepsin G are distinctly different from many other serine proteinases, including fiddler crab collagenase and chymotrypsin. The kcat/Km values obtained for P1 Lys, Phe, Arg and Leu substrates correlate well with those determined for analogous P1 mutants of basic pancreatic trypsin inhibitor (BPTI) obtained through recombinant techniques. To characterise the subsite specificity of the enzyme, a series of Cucurbita maxima trypsin inhibitor I (CMTI I) mutants were used comprising P2-P3' and P12' positions. All the mutants obtained were inhibitors of cathepsin G with association constants in the range: 105-109 M-1. Some of the mutations destabilised complex formation. In particular, Met8-->Arg substitution at P3', which increased association constant for chymotrypsin 46-fold, led to a 7-fold decrease of binding with cathepsin G. In addition, mutation of Ile6 at position P1' either to Val or Asp was deleterious for cathepsin G. In two cases (Ala18-->Gly (P12') and Pro4-->Thr (P2)), about a 10-fold increase in association constants was observed.
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PMID:Specificity of human cathepsin G. 967 78

Neutrophil collagenase or collagenase 2 (MMP-8) is unique among the family of matrix metalloproteinases (MMPs) because of its exclusive pattern of expression in inflammatory conditions. At present, no evidence of the occurrence of this enzyme in tissues other than human has been reported. In this work, we have cloned the murine homologue of human collagenase 2. The isolated cDNA contains an open reading frame coding for a polypeptide of 465 amino acids, which is 74% identical to its human counterpart. The mouse collagenase 2 exhibits the domain structure characteristic of several MMPs, including a signal sequence, a prodomain with the cysteine residue essential for enzyme latency, an activation locus with the Zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. It also contains the three conserved residues (Tyr-209, Asp-230, and Gly-232) located around the Zinc-binding site and are distinctive of the collagenase subfamily. Northern blot analysis of RNAs isolated from a variety of mouse tissues revealed that collagenase 2 is expressed at late stages during mouse embryogenesis, coinciding with the appearance of hematopoietic cells. In addition, collagenase 2 was highly expressed in the postpartum uterus starting at 1 day postpartum and extending up to 5 days. Enzymatic analysis revealed that matrilysin, another MMP overexpressed in uterine tissue, is able to activate murine procollagenase 2. These data suggest that both enzymes could form an activation cascade resulting in the generation of the collagenolytic activity required during the process of massive connective tissue resumption occurring in the involuting uterus.
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PMID:Collagenase 2 (MMP-8) expression in murine tissue-remodeling processes. Analysis of its potential role in postpartum involution of the uterus. 972 11


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