The role of plasminogen activators in the regulation of connective tissue metalloproteinases.
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Animals↗
Biomedical subjects
G Murphy
Publications and source records attributed to G Murphy.
Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases.
The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely.
Animals↗
Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.
The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.
Amino Acid Sequence↗
The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.
The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.
Aggrecans↗
Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.
The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.
Amino Acid Sequence↗
The role of the C-terminal domain in collagenase and stromelysin specificity.
Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.
Amino Acid Sequence↗
The matrix metalloprotease matrilysin (PUMP) is expressed in developing human mononuclear phagocytes.
Matrilysin (PUMP, MMP-7) is a member of the metalloprotease gene family, whose constituents are responsible for the remodeling of extracellular matrix. The matrilysin protein is a 28-kDa zymogen possessing catalytic activities against a broad range of extracellular matrix substrates including proteoglycans, gelatin, fibronectin, laminin, and elastin. To gain insights into the biological expression of matrilysin in human cell types, we generated a monospecific, polyclonal antibody against a 16-amino acid sequence derived from its catalytic domain, a region which lacked significant homology with other matrix metalloenzymes. We found this antibody capable of precipitating a 28-kDa protein from the conditioned media of human bone marrow-derived promonocytes and human peripheral blood monocytes cultivated in vitro. Promonocyte matrilysin was rapidly converted to a 19-kDa form by organomercurial activation. While matrilysin was constitutively synthesized by bone marrow-derived promonocytes, its secretion was markedly up-regulated by the mononuclear phagocyte activator, lipopolysaccharide. Furthermore, despite its expression in monocyte precursors, blood monocytes, and monocyte-derived macrophages, matrilysin was not synthesized by human alveolar macrophages under any tested condition. In situ hybridization studies with matrilysin cRNA confirmed the presence of specific mRNA in both human promonocytes and monocytes. Moreover, a marked increase in hybridizable mRNA was observed with lipopolysaccharide treatment suggesting that matrilysin synthesis is pretranslationally regulated. In summary, this represents the first report documenting constitutive and regulated synthesis of matrilysin by a normal human cell type and suggests that matrilysin is expressed as a significant secreted product of mononuclear phagocytes at an intermediate stage of cellular differentiation.
Amino Acid Sequence↗
The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases.
Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.
Base Sequence↗
A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.
(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.
Amino Acid Sequence↗
The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases.
Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.
Amino Acid Sequence↗
Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A.
The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.
Base Sequence↗
The transoral approach to the cervical spine.
The transoral surgical approach is useful for operating on structures at the base of the brain and the upper cervical spinal cord. For example, this route has been used for resecting spinal tumors and clipping vertebrobasilar aneurysms. In the past, this surgical approach was not advocated due to concerns about exposure and infection. However, the availability of the microscope, computed tomography, computed myelotomography, magnetic resonance imaging and intraoperative radiography as well as more effective techniques have improved the diagnosis of pathology of the craniovertebral junction and surgical performance. An understanding of the operative procedures involved with this approach assists the neuro-science nurse in preoperative teaching and anticipating potential postoperative complications.
Arthritis, Rheumatoid↗
Unilateral linear capillaritis.
We present four cases of a distinctive type of pigmented purpuric eruption occurring in a striking linear and pseudo-dermatomal distribution in young males. Whilst these cases share some of the clinical and histological features of other pigmented purpuric dermatoses, they are not readily classified with any of the entities so far defined in this group of disorders. We believe these cases represent a distinct group not previously described. Four patients with an unusual pigmented purpuric eruption are presented.
Adolescent↗
Rates of caesarean section and neonatal mortality.
A comparison of the yearly statistics of National Maternity Hospital (Dublin) and University Hospital (Newark) was undertaken for the years 1983-1989. The findings appear to indicate that after the elimination of major confounding factors, the substantially higher rates of Caesarean section in Newark (17.5% versus 5.8%) did not bring about a measurable reduction in the rate of neonatal losses. The impact of paediatric care upon the respective neonatal outcomes could not be assessed on the ground of the reviewed data.
Cesarean Section↗
Molecular characterization and regulation of the rhizosphere-expressed genes rhiABCR that can influence nodulation by Rhizobium leguminosarum biovar viciae.
A group of four rhi (rhizosphere-expressed) genes from the symbiotic plasmid of Rhizobium leguminosarum biovar viciae has been characterized. Although mutation of the rhi genes does not normally affect nodulation, in the absence of the closely linked nodulation genes nodFEL, mutations in the rhi genes can influence the nodulation of the vetch Vicia hirsuta. The DNA sequence of the rhi gene region reveals four large open reading frames, three of them constituting an operon (rhiABC) transcribed convergently toward the fourth gene, rhiR. rhiABC are under the positive control of RhiR, the expression of which is repressed by flavonoids that normally induce nod gene expression. This repression, which requires the nodD gene product (the transcriptional activator of nod gene expression), may be due to a cis effect caused by a high level of NodD-dependent expression from the adjacent nodO promoter, which is transcribed divergently from rhiR. RhiR shows significant similarities to a subfamily of transcriptional regulators that includes the LuxR and UvrC-28K proteins. RhiA shows limited homology to a short domain of the lactose permease, LacY, close to a region thought to be involved in substrate binding. No strong homologies were found for the other rhi gene products. It appears that RhiA and RhiB are cytoplasmic, whereas RhiC is a periplasmic protein, since it has a typical N-terminal transit sequence and a rhiC-phoA protein fusion expresses alkaline phosphatase activity. The biochemical role of the rhi genes has not been established, but it appears that they may play a role in the plant-microbe interaction, possibly by allowing the bacteria to metabolize a plant-made metabolite.
Amino Acid Sequence↗
Inhibition of human gall bladder mucus synthesis in patients undergoing cholecystectomy.
Hypersection of gall bladder mucus is associated with gall stone formation in animal models. Aspirin inhibits both mucus synthesis and secretion, prevents gall stone formation in animals and reduces gall stone recurrence in man after dissolution therapy. Mucus biosynthesis in human gall bladder mucosal explants is inhibited by aspirin in vitro. We have studied the effects of aspirin in vivo. Fifty five patients with functioning gall bladder and stones have been randomised, 27 to group 1 (aspirin EC 300 mg once daily for seven days before cholecystectomy) and 28 to group 2 (controls). Gall bladder bile composition was analysed and mucus synthesis rates measured using 3H-glucosamine incorporation into mucosal explants cultured for 24 hours. Patient age, sex, and gall bladder histology were similar in both groups. There were no differences in stone composition, gall bladder bile calcium concentration, cholesterol saturation and cholesterol nucleation time. The mean 3H-glucosamine incorporation in aspirin treated patients was 1347 fmol/g wet weight as compared with 2008 fmol/g wet weight in controls (95% confidence interval 222-1100, p<0.005, unpaired t test). This reduction in biosynthesis was associated with gall bladder bile mucus concentrations of 7.6 mg/ml in patients and 7.1 mg/ml in controls (ns). Treatment with aspirin led to a significant reduction in mucus biosynthesis by the gall bladder mucosa. This action is consistent with a role for aspirin in the prevention of gall stones.
Adult↗
The matrix metalloproteinases and their inhibitors.
A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.
Amino Acid Sequence↗
Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver.
Hepatic lipocytes play a central role in the pathogenesis of liver fibrosis, both via production of extracellular matrix proteins and through secretion of matrix metalloproteinases. In this study, we have characterized lipocyte expression and release of tissue inhibitor of metalloproteinases-1 (TIMP-1), an important inhibitor of metalloproteinase activity, whose role in liver has not previously been examined. TIMP-1 was immunolocalized to human lipocytes, and secretion of TIMP-1 was confirmed by ELISA of culture media; (mean +/- SD) 159 +/- 79 ng of TIMP-1/10(6) cells per 24 h. Evidence for functional inhibitory activity of released TIMP-1 was obtained by (a) reverse zymography that demonstrated a single inhibitor band, M(r) 28 kD, that co-migrated with a TIMP-1-positive control sample; and (b) unmasking of inhibited gelatinase activity in lipocyte medium by separating it from TIMP-1 using gelatin sepharose chromatography; gelatinase activity in chromatographed medium increased more than 20-fold, compared with unfractionated medium, and could be reinhibited by adding back fractions that contained inhibitor. By Northern analysis, freshly isolated human lipocytes exhibited low levels of mRNA expression for TIMP-1, but this increased markedly relative to beta-actin expression with lipocyte activation during cell culture. We conclude that human hepatic lipocytes synthesize TIMP-1, a potent metalloproteinase inhibitor, and that TIMP-1 expression increases with lipocyte activation. These data indicate that hepatic lipocytes can regulate matrix degradation in the liver, and suggest that expression of TIMP-1 by activated lipocytes may contribute to the progression of liver fibrosis.
Adipose Tissue↗