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Overexpression of specific matrix metalloproteinases (MMPs) has a key role in development of several diseases, such as cancer, neurological disorders, and cardiovascular diseases due to their critical role in degradation and remodeling of the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs), a family of four in humans, are endogenous inhibitors of MMPs. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible for inhibition of MMPs to develop efficient therapeutics targeting specific MMPs. We used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library in yeast to identify the dominant minimal MMP inhibitory regions. The minimal TIMP variants screened toward MMP-3 and MMP-9 using fluorescent-activated cell sorting (FACS). Interestingly, several minimal TIMP variants selected after screening toward MMP-3cd or MMP-9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP-3 and MMP-9. The TIMP-MMP binding dissociation constant (K D ), in the nM range, and MMP inhibition constants (K i ), in the pM range, of these minimal TIMP variants were similar to the N-terminal domain of TIMP-1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular docking of the minimal TIMP variants in complex with MMP-3cd to understand the binding and inhibition mechanism of these variants., (© 2023 The Protein Society.)

Protease inhibitors are of considerable interest as anticancer agents. Matrix metalloproteinases (MMPs) were the earliest type of proteases considered as anticancer targets. The developments of MMP inhibitors (MMPIs) by pharmaceutical companies can be dated from the early 1980s. Thus far, none of the over 50 MMPIs entering clinical trials have been approved. This work summarizes the reported studies on the structure of MMPs and complexes with ligands and inhibitors, based on which, the authors analyzed the clinical failures of MMPIs in a structural biological manner. Furthermore, MMPs were systematically compared with urokinase, a protease-generating plasmin, which plays similar pathological roles in cancer development; the reasons for the clinical successes of urokinase inhibitors and the clinical failures of MMPIs are discussed.

Matrix metalloproteinases are enzymes that degrade the extracellular matrix. They have different substrates but similar structural organization. Matrix metalloproteinases are involved in many physiological and pathological processes and there is a need to develop inhibitors for these enzymes in order to modulate the degradation of the extracellular matrix (ECM). There exist two classes of inhibitors: endogenous and synthetics. The development of synthetic inhibitors remains a great challenge due to the low selectivity and specificity, side effects in clinical trials, and instability. An extensive review of currently reported synthetic inhibitors and description of their properties is presented., Competing Interests: The authors declare no conflict of interest.

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729-Val-730 and Glu-732-Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β-binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1-binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor., (© 2019 Colige et al.)

De novo drug discovery is still a challenge in the search for potent and selective modulators of therapeutically relevant target proteins. Here, we disclose the unexpected discovery of a peptidic ligand 1 by X-ray crystallography, which was auto-tailored by the therapeutic target MMP-13 through partial self-degradation and subsequent structure-based optimization to a highly potent and selective β-sheet peptidomimetic inhibitor derived from the endogenous tissue inhibitors of metalloproteinases (TIMPs). The incorporation of non-proteinogenic amino acids in combination with a cyclization strategy proved to be key for the de novo design of TIMP peptidomimetics. The optimized cyclic peptide 4 (ZHAWOC7726) is membrane permeable with an IC 50 of 21 nm for MMP-13 and an attractive selectivity profile with respect to a polypharmacology approach including the anticancer targets MMP-2 (IC 50 : 170 nm) and MMP-9 (IC 50 : 140 nm)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology.

Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys³⁰ responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of Tg-TIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4-like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Remodeling of the myocardium and the extracellular matrix (ECM) occurs in heart failure irrespective of its initial cause. The ECM serves as a scaffold to provide structural support as well as housing a number of cytokines and growth factors. Hence, disruption of the ECM will result in structural instability as well as activation of a number of signaling pathways that could lead to fibrosis, hypertrophy, and apoptosis. The ECM is a dynamic entity that undergoes constant turnover, and the integrity of its network structure is maintained by a balance in the function of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). In heart disease, levels of MMPs and TIMPs are altered resulting in an imbalance between these two families of proteins. In this review, we will discuss the structure, function, and regulation of TIMPs, their MMP-independent functions, and their role in heart failure. We will review the knowledge that we have gained from clinical studies and animal models on the contribution of TIMPs in the development and progression of heart disease. We will further discuss how ECM molecules and regulatory genes can be used as biomarkers of disease in heart failure patients.

Orchestration of the growth and remodeling of tissues and responses of cells to their extracellular environment is mediated by metalloproteinases of the Metzincin clan. This group of proteins comprises several families of endopeptidases in which a zinc atom is liganded at the catalytic site to three histidine residues and an invariant methionine residue. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinase (MMPs) family, and also of families such as the disintegrin metalloproteinases (ADAM and ADAMTS). TIMPs therefore have a pivotal role in determining the influence of the extracellular matrix, of cell adhesion molecules, and of many cytokines, chemokines and growth factors on cell phenotype. The TIMP family is an ancient one, with a single representative in lower eukaryotes and four members in mammals. Although much is known about their mechanism of action in proteinase regulation in mammalian cells, less is known about their functions in lower organisms. Recently, non-inhibitory functions of TIMPs have been identified in mammalian cells, including signaling roles downstream of specific receptors. There are clearly still questions to be answered with regard to their overall roles in biology.

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom and the human genome contains four paralogous genes encoding TIMPs 1 to 4. TIMPs were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has now been found to be broader as it includes the inhibition of several of the disintegrin-metalloproteinases, ADAMs and ADAMTSs. TIMPs are therefore key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. Structural studies of TIMP-MMP complexes have elucidated the inhibition mechanism of TIMPs and the multiple sites through which they interact with target enzymes, allowing the generation of TIMP variants that selectively inhibit different groups of metalloproteinases. Engineering such variants is complicated by the fact that TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. Receptors responsible for some of these activities have been identified and their signaling pathways have been investigated. A series of studies using mice with specific TIMP gene deletions has illuminated the importance of these molecules in biology and pathology., (Copyright 2010 Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) are a group of proteases that belong to the metazincin family. These proteins consist of similar structures featuring a signaling peptide, a propeptide domain, a catalytic domain where the notable zinc ion binding site is found and a hinge region that binds to the C-terminal hemoplexin domain. MMPs can be produced by numerous cell types through secretion or localization to the cell membrane. While certain chemical compounds have been known to generally inhibit MMPs, naturally occurring proteins known as tissue inhibitors of metalloproteinases (TIMPs) effectively interact with MMPs to modify their biological roles. MMPs are very important enzymes that actively participate in remodeling the extracellular matrix by degrading certain constituents, along with promoting cell proliferation, migration, differentiation, apoptosis and angiogenesis. In normal adult tissue, they are almost undetectable; however, when perturbed through injury, disease or pregnancy, they have elevated expression. The goal of this review is to identify new experimental findings that have provided further insight into the role of MMPs in skeletal muscle, nerve and dermal tissue, as well as in the liver, heart and kidneys. Increased expression of MMPs can improve the regeneration potential of wounds; however, an imbalance between MMP and TIMP expression can prove to be destructive for afflicted tissues.

Ac-TMP-2, an immunodominant hookworm antigen encoding a tissue inhibitor of metalloproteinase (TIMP) was cloned by immunoscreening an Ancylostoma caninum larval cDNA library with sera pooled from dogs immunized with irradiated A. caninum third stage larvae (ir-L3). The open reading frame of Ac-tmp-2 cDNA encoded a 244 amino acids (predicted molecular weight of 27.7 kDa), which shared a common N-terminus with other vertebrate and invertebrate TIMPs, including Ac-TMP-1, the most abundant adult hookworm secreted protein. However Ac-TMP-2 also contains an unusual multicopy (ten) repeat of the amino acid sequence, KTVEENDE. By immunoblotting, Ac-TMP-2 was detected only in adult hookworms and their excretory secretory products although the corresponding mRNA was also detected in L3. Immunolocalization with specific antiserum showed that native Ac-TMP-2 was located in adult worm's esophagus and cephalic glands. Recombinant Ac-TMP-2 expressed in bacteria was highly immunogenic and recognized by ir-L3 immunized dog immune sera. The recombinant Ac-TMP-2 protein inhibited the human matrix metalloproteinases, MMP-2, MMP-7 and MMP-13. As an immunodominant protein having a possible role in the parasite-host relationship of canine hookworm infection, recombinant Ac-TMP-2 represents a plausible target for vaccine development.

Tissue inhibitors of metalloproteinases (TIMPs) regulate diverse processes, including extracellular matrix (ECM) remodeling, and growth factors and their receptors' activities through the inhibition of matrix metalloproteinases (MMPs). Recent evidence has shown that this family of four members (TIMP-1 to TIMP-4) can also control other important processes, such as proliferation and apoptosis, by a mechanism independent of their MMP inhibitory actions. Of these inhibitors, the most recently identified and least studied is TIMP-4. Initially cloned in human and, later, in mouse, TIMP-4 expression is restricted to heart, kidney, pancreas, colon, testes, brain and adipose tissue. This restricted expression suggests specific and different physiological functions. The present review summarizes the information available for this protein and also provides a putative structural model in order to propose potential relevant directions toward solving its function and role in diseases such as cancer.

Matrix metalloproteinases (MMPs) are now acknowledged as key players in the regulation of both cell-cell and cell-extracellular matrix interactions. They are involved in modifying matrix structure, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury and in the progression of diseases such as arthritis, cancer and cardiovascular disease. Because of their wide spectrum of activities and expression sites, the elucidation of their potential as drug targets in disease or as important features of the repair process will be dependent upon careful analysis of their role in different cellular locations and at different disease stages. Novel approaches to the specific regulation of individual MMPs in different contexts are also being developed.

Matrix metalloproteinases (MMPs) belong to a large family of multidomain zinc endopeptidases. They are one of the most important proteolitic enzymes which digest components of the extracellular matrix and abundant macromolecules on cell surface and take part in many physiological processes, such as apoptosis or angiogenesis. MMPs are also engaged in the pathogenesis of many diseases such as arthritis and cancer. The development of effective inhibitors and discovery of their mechanisms of action can have significant influence on therapeutic strategy.

The backbone mobility of the C-terminal domain of procollagen C-proteinase enhancer (NTR PCOLCE1), part of a connective tissue glycoprotein, was determined using 15N NMR spectroscopy. NTR PCOLCE1 has been shown to be a netrin-like domain and adopts an OB-fold such as that found in the N-terminal domain of tissue inhibitors of metalloproteinases-1 (N-TIMP-1), N-TIMP-2, the laminin-binding domain of agrin and the C-terminal domain of complement protein C5. NMR relaxation dynamics of NTR PCOLCE1 highlight conformational flexibility in the N-terminus, strand A and the proximal CD loop. This region in N-TIMP is known to be essential for inhibitory activity against the matrix metalloproteinases and suggests that this region is of equal importance for NTR PCOLCE1, although the specific functional activity of the NTR PCOLCE1 domain is still unknown. Dynamics observed within the structural core of NTR PCOLCE1 that are not observed in N-TIMP molecules suggest that although the two domains have a similar architecture, the NTR PCOLCE1 domain will show different thermodynamic properties on binding and hence the target molecule could be somewhat different from that observed for the TIMPs. ModelFree order parameters show that NTR PCOLCE1 has more flexibility than both N-TIMP-1 and N-TIMP-2.

Peroxynitrite, via post-translational modifications to target proteins, contributes to cardiovascular injury and cancer. Since tissue inhibitor of metalloproteinase-4 (TIMP-4), the activity of which is impaired in both pathological conditions, has several amino acid residues susceptible to peroxynitrite, we investigated its role as a potential target of peroxynitrite. Peroxynitrite-induced nitration and oligomerization of TIMP-4 attenuated its inhibitory activity against MMP-2 activity and endothelial or tumor cell invasiveness. Moreover, cell treatment with peroxynitrite promoted the nitration of endogenous TIMP-4. HPLC/ESI-MS/MS analysis of peroxynitrite-treated TIMP-4 showed modifications at Y114, Y195, Y188 and Y190. In conclusion, TIMP-4 nitration might be a potential mechanism contributing to cardiovascular disease and cancer.

The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production can be stimulated by the hormone relaxin. This hormone facilitates delivery by softening the connective tissues of the reproductive tract, and it prepares the mammary gland for lactation. Periodontal remodelling takes place during orthodontic tooth movement, which might be enhanced by relaxin. Therefore, we investigated the effects of relaxin on gelatinase expression of human PDL cells. Cultures of human PDL cells were incubated with relaxin. Gelatinase (MMP-2 and -9) expression, alpha-smooth muscle actin expression (alpha-SMA), total MMP activity and DNA content were measured. Both proMMP-2 and active MMP-2 was identified in the cultures. There was a clear trend showing a dose-dependent increase of MMP-2 production, which was significant at 250 ng/ml. Total MMP activity was not affected. A stimulation of alpha-SMA expression was found at 50 ng/ml. The results indicate that relaxin activates human PDL cells by the stimulation of MMP-2 and alpha-smooth muscle actin.

Matrix metalloproteinase 13 (MMP13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis. Clinical administration of broad spectrum MMP inhibitors such as marimastat has been implicated in severe musculo-skeletal side effects. Consequently, research has been focused on designing inhibitors that selectively inhibit MMP13, thereby circumventing musculo-skeletal toxicities. A series of pyrimidine dicarboxamides were recently shown to be highly selective inhibitors of MMP13 with a novel binding mode. We have applied a molecular ruler to this exosite by dual inhibition studies involving a potent dicarboxamide in the presence of two metal chelators of different sizes. A larger hydroxamate mimic overlaps and antagonizes binding of the dicarboxamide to the exosite whereas the much smaller acetohydroxamate synergizes with the dicarboxamide. These studies elucidate the steric requirement for compounds that fit exclusively into the active site, a mandate for generating highly selective MMP13 inhibitors.

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.

Matrix metalloproteinases (MMPs) are a large family of proteinases that remodel extracellular matrix (ECM) components and cleave a number of cell surface proteins. MMP activity is regulated via a number of mechanisms, including inhibition by tissue inhibitors of metalloproteinases (TIMPs). Originally thought to cleave only ECM proteins, MMP substrates are now known to include signaling molecules (growth factor receptors) and cell adhesion molecules. Recent data suggest a role for MMPs in a number of renal pathophysiologies, both acute and chronic. This review will focus on the expression and localization of MMPs and TIMPs in the kidney, as well as summarizing the current information linking these proteins to acute kidney injury, glomerulosclerosis/tubulointerstitial fibrosis, chronic allograft nephropathy, diabetic nephropathy, polycystic kidney disease, and renal cell carcinoma.

This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.

This chapter provides practical information on the assay of tissue inhibitor of metalloproteinase (TIMP) activity and background information enabling meaningful interpretation of the data. Protocols are given for assessing the presence of TIMPs in biological samples by immunoblotting and by virtue of their ability to inhibit matrix metalloproteinase (MMP) hydrolysis of protein substrates (reverse zymography) and synthetic fluorogenic substrates.

Protein-protein interactions are governed by a variety of structural features. The sequence specificities of such interactions are usually easier to establish than the "topological specificities," whereby interactions may be classified based on recognition of distinct three-dimensional structural motifs. Approaches to explore topological specificities have been based primarily on assembly of mini-proteins with well defined secondary, tertiary, and/or quarternary structures. The present chapter focuses on three approaches for constructing topologically well defined mini-proteins: template-assembled synthetic proteins (TASPs), disulfide-stabilized structures, and peptide-amphiphiles (PAs). Specific examples are given for applying each approach to explore topologically-dependent protein-protein interactions. TASPs are utilized to identify a metastatic melanoma receptor that binds to the alpha1(IV)1263-1277 region of basement membrane (type IV) collagen. A disulfide-stabilized structure incorporating a sarafotoxin (SRT) 6b model was examined as a matrix metalloproteinase (MMP)-3 inhibitor. PAs were developed as (a) fluorogenic triple-helical or polyPro II substrates for MMPs and aggrecanase members of the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and (b) glycosylated and nonglycosylated ligands for metastatic melanoma cells. Topologically constrained mini-proteins have proved to be quite versatile, helping to define critical primary, secondary, and tertiary structural elements that modulate enzyme and receptor functions.

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3-MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.

A series of N-benzoyl 4-aminobutyric acid hydroxamate analogs were synthesized and evaluated as matrix metalloproteinase inhibitors. Synthetic work was focused on the chemical modification of the 4-aminobutyric acid part using easily available starting materials. As such, chemical modification was carried out using commercially available starting materials such as 4-aminobutyric acid, (+)- and (-)-malic acid, and D- and L-glutamic acid derivatives. Among the compounds tested, N-[4-(benzofuran-2-yl)benzoyl] 4-amino-4S-hydroxymethylbutyric acid hydroxamates derived from L-glutamic acid demonstrated more potent inhibitory activity against MMP-2 and MMP-9 compared with the corresponding 2S-hydroxy analogs or 3S-hydroxy analogs, respectively, which were derived from (-)-malic acid. Structure-activity relationship study is presented.

Matrix metalloproteinases (MMPs), also called matrixins, function in the extracellular environment of cells and degrade both matrix and non-matrix proteins. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury, e.g. after myocardial infarction, and in progression of diseases such as atheroma, arthritis, cancer and chronic tissue ulcers. They are multi-domain proteins and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). This review introduces the members of the MMP family and discusses their domain structure and function, proenyme activation, the mechanism of inhibition by TIMPs and their significance in physiology and pathology.

Matrix Metalloproteinases (MMPs) are a family of multidomain zinc endopeptidases that function in the extracellular space or attached to the cell membrane. Their proteolytic activity is controlled by the presence of endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-macroglobulin and others. Disruption of the proteinase-inhibitor balance is observed in serious diseases such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for drug intervention by pharmacological inhibitors. The determination of MMP structures is of critical importance in order to understand their substrate preferences, dimerization events, and their association with matrix components and inhibitors. Thus, MMP structures may contribute significantly to the development of specific MMP inhibitors, which should allow precise control of individual members of the MMP family without affecting all members or the closely related metalloproteinases such as ADAMs and ADAMTSs.

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will focus on zymographical techniques for the analysis of MMPs and TIMPs. MMPs can be analyzed with several zymographical techniques, but substrate zymography is the most commonly used. This technique identifies MMPs by the degradation of their preferential substrate and by their molecular weight. Several substrates that can be used for zymography are described. Reverse zymography, which detects TIMPs by their ability to inhibit MMPs, is also discussed. Finally, in situ zymography is described, which is used to localize MMPs in tissue sections. Common problems encountered during sample preparation, zymography itself and the data analysis are discussed. Hints are given to improve the sensitivity and accuracy of zymographical methods. In conclusion, zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression.

This review describes the clinical status (based on available information) of experimental drugs that inhibit enzymes called proteases, or more precisely a sub-class of proteases called peptidases that catalyse the hydrolysis of polypeptide main chain amide bonds. These peptidases are classified by the key catalytic residue in the active site of the enzyme that effects hydrolysis, namely aspartic, serine, cysteine, metallo or threonine proteases. In this review we show structures for 108 inhibitors of these enzymes and update the clinical disposition of over 100 inhibitors that have been considered worthy enough by pharmaceutical, biotechnology or academic researchers and their financial backers to be trialed in humans as prospective medicines. We outline some of their chemical and pharmacological characteristics and compare the current status of protease inhibitors in the clinic with what was observed about 5 years ago (Leung et al, J. Med. Chem. 2000, 43, 305-341). We assess the progress of protease inhibitors into man, predict their futures, and outline some of the hurdles that have been overcome and that still remain for this promising class of new therapeutic agents.

Our previously developed approach to the development of QSAR equations for benzene derivatives, originally for phenylalkylamine hallucinogens, has been applied to four new systems: sulfonamide inhibitors of the enzymes carbonic anhydrase, thrombin, trypsin, and Clostridium histolyticum collagenase. The novel features involve the energies and nodal orientations of pi-like orbitals, and an allowance for the symmetry of the benzene nucleus. The resulting equations give better fits, better predictivity and are more easily interpretable than those resulting from traditional QSAR methods.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs) found in most tissues and body fluids. By inhibiting MMPs activities, they participate in tissue remodeling of the extracellular matrix (ECM). The balance between MMPs and TIMPs activities is involved in both normal and pathological events such as wound healing, tissue remodeling, angiogenesis, invasion, tumorigenesis and metastasis. The intracellular signalling controlling both TIMPs and MMPs expression begins to be elucidated and gaining insights into the molecular mechanisms regulated by TIMPs and MMPs could represent a new approach in the development of potential therapeutics. Numerous investigations have pointed out that TIMPs exhibit multifunctional activities distinct from MMP inhibition. In this review, we detailed the multiple activities of TIMPs in vivo and in vitro and we reported their implication in physiological and pathological processes. Further, we documented recent studies of their role in hematopoiesis and we itemized the different signalling pathways they induced.

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.

The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals. The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs). In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro. The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs. Titration with the catalytic domain of human MMP-3 [MMP-3(DeltaC)] showed that dN-TIMP prepared by this method is correctly folded and fully active. dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs. dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10. Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity. Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.

Fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). There are unique interactions between TIMP-2 and MT1-MMP, which we have previously defined, and here we identify TIMP-2 sequence motifs specific for proMMP-2 binding in the context of its activation by MT1-MMP. A TIMP-2 mutant encoding the C-terminal domain of TIMP-4 showed loss of proMMP-2 activation, indicating that the C-terminal domain of TIMP-2 is important in establishing the trimolecular complex between MT1-MMP, TIMP-2 and proMMP-2. This was confirmed by analysis of a TIMP-4 mutant encoding the C-terminal domain of TIMP-2, which formed a trimolecular complex and promoted proMMP-2 processing to the intermediate form. Mutants encoding TIMP-4 from Cys(1) to Leu(185) and partial tail sequence of TIMP-2 showed some gain of activating capability relative to TIMP-4. The identified residues were subsequently mutated in TIMP-2 (E(192)-D(193) to I(192)-Q(193)) and this inhibitor showed a significantly reduced ability to facilitate proMMP-2 processing by MT1-MMP. Furthermore, the tail-deletion mutant Delta(186-194)TIMP-2 was completely incapable of promoting proMMP-2 activation by MT1-MMP. Thus the C-terminal tail residues of TIMP-2 are important determinants for stable trimolecular complex formation between TIMP-2, proMMP-2 and MT1-MMP and play an important role in MT1-MMP-mediated processing to the intermediate and final active forms of MMP-2 at the cell surface.

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.

Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases play a central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration. Currently 23 MMP genes have been identified in humans, and most are multidomain proteins. This review describes the members of the matrixin family and discusses substrate specificity, domain structure and function, the activation of proMMPs, the regulation of matrixin activity by tissue inhibitors of metalloproteinases, and their pathophysiological implication.

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.

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.

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k(on) rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by approximately 2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by approximately 75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu(192) and Asp(193) in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.

There are currently 25 known vertebrate matrix metalloproteinases (MMPs) and 4 tissue inhibitors of metalloproteinases (TIMPs). This article reviews these proteases from an historical perspective in terms of who discovered each protein, when the sequence was established, when action on protein substrates was demonstrated, and what names have been used. A similar approach is taken for the TIMPS, and their multiple functions in addition to protease inhibition are emphasized. MMPs from invertebrates, plants, and bacteria are also discussed. This review is an outgrowth and update of a chapter by the same name originally published in Matrix Metalloproteinase Protocols, pp. 1-23, edited by I. M. Clark and published by Humana Press in 2001.