Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs), enzymes that play central roles in the degradation of extracellular matrix components. The balance between MMPs and TIMPs is important in the maintenance of tissues, and its disruption affects tissue homoeostasis. Four related TIMPs (TIMP-1 to TIMP-4) can each form a complex with MMPs in a 1:1 stoichiometry with high affinity, but their inhibitory activities towards different MMPs are not particularly selective. The three-dimensional structures of TIMP-MMP complexes reveal that TIMPs have an extended ridge structure that slots into the active site of MMPs. Mutation of three separate residues in the ridge, at positions 2, 4 and 68 in the amino acid sequence of the N-terminal inhibitory domain of TIMP-1 (N-TIMP-1), separately and in combination has produced N-TIMP-1 variants with higher binding affinity and specificity for individual MMPs. TIMP-3 is unique in that it inhibits not only MMPs, but also several ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin motifs) metalloproteinases. Inhibition of the latter groups of metalloproteinases, as exemplified with ADAMTS-4 (aggrecanase 1), requires additional structural elements in TIMP-3 that have not yet been identified. Knowledge of the structural basis of the inhibitory action of TIMPs will facilitate the design of selective TIMP variants for investigating the biological roles of specific MMPs and for developing therapeutic interventions for MMP-associated diseases.     

Biophysical and functional characterization of full-length, recombinant human tissue inhibitor of metalloproteinases-2 (TIMP-2) produced in Escherichia coli. Comparison of wild type and amino-terminal alanine appended variant with implications for the mechanism of TIMP functions.

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.     

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.     

Cloning and developmental regulation of tissue inhibitor of metalloproteinases-3 (TIMP3) in Xenopus laevis early embryos

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn¹⁸⁴, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Post-translational proteolytic processing of procollagen C-terminal proteinase enhancer releases a metalloproteinase inhibitor

Activity of matrix metalloproteinases (MMP) is regulated by a family of proteins called tissue inhibitors of metalloproteinases (TIMP). Four TIMPs have been cloned, and their molecular weights range from 29,000 to 20,000. By reverse zymography, we have observed a metalloproteinase inhibitor with an apparent molecular weight of 16, 500 from medium conditioned by human brain tumor cells. Antibodies directed against TIMPs failed to react with the 16,500 molecular weight inhibitor, indicating that it was not a truncated form of a known TIMP. The inhibitor was isolated from conditioned medium using affinity and ion exchange chromatography. N-terminal sequences of the inhibitor matched amino acid sequences within the C-terminal domain of a protein known as procollagen C-terminal proteinase enhancer (PCPE). Thus, the inhibitor was named CT-PCPE. Comparison of the N-terminal domain of TIMP with CT-PCPE revealed that both contained six cysteine residues. As in the case of TIMP, reduction and alkylation abolished the inhibitory activity of CT-PCPE. Purified CT-PCPE inhibited MMP-2 with an IC(50) value much greater than that of TIMP-2. This implies that MMPs may not be the physiologic targets for CT-PCPE inhibition. However, these results suggest that CT-PCPE may constitute a new class of metalloproteinase inhibitor.     

Tissue inhibitor of metalloproteinases-4 (TIMP-4) gene expression is increased in human osteoarthritic femoral head cartilage

Tissue inhibitor of metalloproteinases-4 (TIMP-4), the newest member of the TIMP family, blocks the activities of several matrix metalloproteinases (MMPs) implicated in the arthritic cartilage erosion. By utilizing semi-quantitative RT-PCR, immunoblotting, and immunohistochemistry, we investigated whether the TIMP-4 gene is expressed in human non-arthritic and osteoarthritic (OA) cartilage. Directly analyzed femoral head cartilage showed TIMP-4 RNA expression in 2 of 9 non-arthritic and 12 of 14 OA patients. Femoral head cartilage from 6 of 9 OA patients had elevated TIMP-4 protein compared to the low-level expression in 3 of 8 non-arthritic controls. In most patients, there was correlation between TIMP-4 RNA and protein expression. TIMP-4 protein was also detected immunohistochemically in the upper zone of OA cartilage. The widespread TIMP-4 RNA and protein expression and augmentation in femoral OA cartilage suggests its important role in joint tissue remodeling and pathogenesis of OA. Increased TIMP levels in arthritic cartilage may not be a sufficiently effective defense against cartilage resorption by excessive multiple MMPs and aggrecanases.     

Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.     

Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells

An imbalance in the matrix metalloproteinase (MMP) : tissue inhibitor of MMP (TIMP) ratio may be associated with tissue injury. Here, we studied the regulation of TIMP and MMP gene expression in primary glial cultures to ascertain the factors involved in the regulation of these genes in conditions of inflammatory neuropathology. Astrocytes were found to basally express TIMP-1 and TIMP-3 mRNA while microglia expressed only TIMP-2 mRNA. TIMP-4 mRNA was not detectable in either cell type. Treatment with interferon-alpha (IFN-alpha), IFN-gamma, interleukin-3 (IL-3), IL-6 or tumor necrosis factor-alpha (TNF-alpha) did not alter expression of the TIMP genes. However, in astrocytes, but not in microglia, serum, IL-1beta or lipopolysaccharide (LPS) evoked a dose- and time-dependent increase in TIMP-1 mRNA and a coincident down-regulation of the TIMP-3 gene. Astrocytes were found to express mRNA constitutively for MMPs -3, -11 and -14. In contrast, microglia expressed only MMP-12 mRNA under basal conditions. IL-1beta enhanced MMP-3 mRNA levels while LPS increased the MMP-3, -9, -12, -13 and -14 mRNAs. Our findings reveal that regulatory control of TIMP and MMP gene expression by glial cells is agonist- and cell-type specific, and suggest that innate immune signals govern the temporal and spatial expression patterns of TIMP and MMP genes in neuroinflammatory conditions of the CNS.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

Morbillivirus infection of the mouse central nervous system induces region-specific upregulation of MMPs and TIMPs correlated to inflammatory cytokine expression

Viral infection of the central nervous system (CNS) can result in perturbation of cell-to-cell communication involving the extracellular matrix (ECM). ECM integrity is maintained by a dynamic balance between the synthesis and proteolysis of its components, mainly as a result of the action of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). An MMP/TIMP imbalance may be critical in triggering neurological disorders, in particular in virally induced neural disorders. In the present study, a mouse model of brain infection using a neurotropic strain of canine distemper virus (CDV) was used to study the effect of CNS infection on the MMP/TIMP balance and cytokine expression. CDV replicates almost exclusively in neurons and has a unique pattern of expression (cortex, hypothalamus, monoaminergic nuclei, hippocampus, and spinal cord). Here we show that although several mouse brain structures were infected, they exhibited a differential pattern in terms of MMP, TIMP, and cytokine expression, exemplified by (i) a large increase in pro-MMP9 levels, in particular in the hippocampus, which occurred mainly in neurons and was associated with in situ gelatinolytic activity, (ii) specific and significant upregulation of MT1-MMP mRNA expression in the cortex and hypothalamus, (iii) an MMP/TIMP imbalance, suggested by the upregulation of TIMP-1 mRNA in the cortex, hippocampus, and hypothalamus and of TIMP-3 mRNA in the cortex, and (iv) a concomitant region-specific large increase in expression of Th1-like cytokines, such as gamma interferon, tumor necrosis factor alpha, and interleukin 6 (IL-6), contrasting with weaker induction of Th2-like cytokines, such as IL-4 and IL-10. These data indicate that an MMP/TIMP imbalance in specific brain structures, which is tightly associated with a local inflammatory process as shown by the presence of immune infiltrating cells, differentially impairs CNS integrity and may contribute to the multiplicity of late neurological disorders observed in this viral mouse model.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases.

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.     

MMP-TIMP interaction depends on residue 2 in TIMP-4.

Extracellular matrix remodeling and degradation are of great importance in both physiological and pathological situations. Matrix metalloproteinases (MMPs) and their natural occurring inhibitors - tissue inhibitors of metalloproteinases (TIMPs) - are involved in matrix turnover. Among the TIMPs there is only little specificity for inhibiting individual MMPs. In this report we describe the mutational analysis of the interaction of human TIMP-4 with several MMPs. The effects of different substitutions of residue 2 (Ser(2)) in the inhibitory domain of TIMP-4 were determined by kinetic measurements. Size, charge and polarity of residue 2 in the TIMP structure are key factors in MMP inhibition.     

mRNA of bovine tissue inhibitor of metalloproteinase: sequence and expression in bovine ovarian tissue

A cDNA library derived from poly(A+)RNA of bovine ovary was screened with a PCR fragment comprising the coding region of human tissue inhibitor of metalloproteinase inhibitor (TIMP). From a number of positive clones, pBGR19, containing a 747 bp insert, was identified and sequenced. The derived amino acid sequence represents that of the precursor of bovine TIMP. Northern analysis reveals a TIMP specific mRNA of 800 bp. Southern analysis indicates that one gene appears to specify bovine TIMP. TIMP mRNA is only weakly expressed in follicular granulosa- and theca cells, whereas luteinization of the follicle is associated with an increase of expression. Expression varies with the stage of the luteal phase; it was highest in stages I and III, but low in stages II and IV of the oestrous cycle.     

Characterization of C-terminally truncated human tissue inhibitor of metalloproteinases-4 expressed in Pichia pastoris

The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the N-terminal domain of TIMP-4 was analysed. Using a fluorescent MCA-peptide, Ki values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.