Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

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.     

Structural basis of matrix metalloproteinase function

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases which contain a catalytic domain with a common metzincin-like topology. The MMPs are involved not only in extracellular matrix degradation, but also in a number of other biological processes. Normally, their proteolytic activity is regulated precisely 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, tumour 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. Since the first publication of atomic MMP structures in 1994, much more structural information has become available on details of the catalytic domain, on its interaction with synthetic and protein inhibitors, on domain organization and on the formation of complexes with other proteins. This review will outline our current knowledge of MMP structure.     

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.     

Insect inhibitors of metalloproteinases

Two types of peptidic metalloproteinase inhibitors have recently been identified in insects. A homologue of vertebrate tissue inhibitors of metalloproteinases (TIMPs) was found in the fruitfly Drosophila melanogaster which may contributes to regulation of a corresponding matrix metalloproteinase (MMP). The first member of MMPs from insects which shares similarity with vertebrate MMPs has also been cloned and characterized from Drosophila, suggesting conserved evolution of both MMPs and TIMPs. The first insect inhibitor of metalloproteinases (IMPI), which was identified in larvae of the greater wax moth, Galleria mellonella, shares no sequence similarity with known vertebrate or invertebrate proteins and represents the first non-TIMP-like inhibitor of metalloproteinases reported to date. In contrast to TIMPs, the IMPI is not active against MMPs but inhibits microbial metalloproteinases such as bacterial thermolysin. Insects may recognize such toxic metalloproteinases associated with invading pathogens by particular peptidic fragments that result from their nonregulated activity within the hemolymph. Metalloproteinases induce expression of the IMPI along with other antimicrobial proteins in course of humoral immune response of G. mellonella, thereby mediating regulation of metalloproteinase activity released within the hemolymph and inhibition of pathogen development as well.     

Tissue inhibitors of metalloproteinases: evolution, structure and function

The matrix metalloproteinases (MMPs) play a key role in the normal physiology of connective tissue during development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. An important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4). The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Mutations in TIMP-3 are the cause of Sorsby's fundus dystrophy in humans, a disease that results in early onset macular degeneration. This review highlights the evolution of TIMPs, the recently elucidated high-resolution structures of TIMPs and their complexes with metalloproteinases, and the results of mutational and other studies of structure-function relationships that have enhanced our understanding of the mechanism and specificity of the inhibition of MMPs by TIMPs. Several intriguing questions, such as the basis of the multiple biological functions of TIMPs, the kinetics of TIMP-MMP interactions and the differences in binding in some TIMP-metalloproteinase pairs are discussed which, though not fully resolved, serve to illustrate the kind of issues that are important for a full understanding of the interactions between families of molecules.     

Tissue inhibitors of metalloproteinases

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.     

Tissue inhibitors of metalloproteinases

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.     

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases

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.     

Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors

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.     

Engineering of tissue inhibitor of metalloproteinases mutants as potential therapeutics

Matrix metalloproteinases (MMPs) play a central role in many biological processes such as development, morphogenesis and wound healing, but their unbalanced activities are implicated in numerous disease processes such as arthritis, cancer metastasis, atherosclerosis, nephritis and fibrosis. One of the key mechanisms to control MMP activities is inhibition by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs). This review highlights the structures and inhibition mechanism of TIMPs, the biological activities of TIMPs, the unique properties of TIMP-3, and the altered specificity towards MMPs achieved by mutagenesis. A potential therapeutic use of TIMP variants is discussed.     

The molecular biology of matrix metalloproteinases and tissue inhibitors of metalloproteinases in inflammatory bowel diseases

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be predominant proteases and protease inhibitors involved in the pathogenesis of inflammatory bowel diseases (IBD) through their ability to remodel the extracellular matrix (ECM) in response to inflammatory stimuli and by their immunomodulating effects. An imbalance between MMPs and TIMPs has been linked with acute and chronic inflammation and aberrant tissue remodeling, as seen in IBD. Moreover, recurrent phases of tissue destruction and subsequent tissue repair can cause serious complications in IBD patients such as fistulas and fibrosis. The aims of this review are (i) to summarize current literature on genetic association, mRNA, and protein expression studies with regard to MMPs and TIMPs in IBD patients and various animal models, including those with transgenic and knockout mice; (ii) to compare biochemical and molecular biological data in humans with those obtained in animal model studies and (iii) to critically evaluate and translate how this knowledge may be used in practical terms to understand better the pathophysiology and mechanisms operating in IBD and to apply this for improvement of clinical outcomes at diagnostic, prognostic and therapeutic levels.     

Expression profiling of metalloproteinases and their inhibitors in synovium and cartilage

Cartilage destruction in osteoarthritis (OA) is thought to be mediated by two main enzyme families; the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, whereas enzymes from the 'a disintegrin and metalloproteinase domain with thrombospondin motifs' (ADAMTS) family mediate cartilage aggrecan loss. Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of these enzymes. Although cartilage destruction in OA might be driven by the chondrocyte, low-grade synovitis is reported in patients with all grades of this disease.     

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction

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.     

Occurrence and temporal variation in matrix metalloproteinases and their inhibitors during murine secondary palatal morphogenesis

Extracellular matrix (ECM) molecules are known to play a pivotal role in morphogenesis of the secondary palate, and changes in their composition and distribution, not attributable to changes in synthesis, are known to occur during palatogenesis. The present study was undertaken to determine if the enzymes responsible for mediating their degradation, the matrix metalloproteinases (MMP), and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP), are temporospatially regulated during murine palatal shelf morphogenesis. Palatal shelves were harvested at gestational days (gd) 12, 13 and 14. MMPs were identified by gelatin zymography, with and without inhibitors, and the identity of specific bands confirmed by Western blot analysis. TIMPs were identified by reverse zymography. MMP and TIMP messages were detected using RT-PCR with specific primers to MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2. Zymography revealed bands of molecular weights corresponding to MMPs 2, 7, 9 and 13 at all ages examined; the intensity of these bands increased with developmental age. Western blot analysis established the presence of MMP-3 and its developmental variation in expression. RT-PCR demonstrated the presence of mRNA for all MMPs and TIMP at all sampling times and all but MMP-2 showed developmental variation. Whereas increases in mRNA were detected for MMPs 3, 9, and 13, MMP-7 mRNA decreased between gd 12 and 14. The results of this study demonstrate that MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2 and their messages are present during the course of palatal shelf remodelling and that their expression is temporally regulated.     

Heterogeneous effects of tissue inhibitors of matrix metalloproteinases on cardiac fibroblasts

The balance between matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), plays a critical role in cardiac remodeling. Although a number of studies have characterized the pathophysiological role of MMPs in the heart, very little is known with respect to the role of TIMPs in the heart. To delineate the role of TIMPs in the heart we examined the effects of adenovirus-mediated overexpression of TIMP-1, -2, -3, and -4 in cardiac fibroblasts. Infection of cardiac fibroblasts with adenoviral constructs containing human recombinant TIMP (AdTIMP-1, -2, -3, and -4) provoked a significant (P < 0.0001) 1.3-fold in increase in bromodeoxyuridine (BrdU) incorporation. Similarly, treatment of cardiac fibroblasts with AdTIMP-1-, -2-, -3-, and -4-conditioned medium led to a 1.2-fold increase in BrdU incorporation (P < 0.0001) that was abolished by pretreatment with anti-TIMP-1, -2, -3, and -4 antibodies. The effects of TIMPs were not mimicked by treating the cells with RS-130830, a broad-based MMP inhibitor, suggesting that the effects of TIMPs were independent of their ability to inhibit MMPs. Infection with AdTIMP-1, -2, -3, and -4 led to a significant increase in alpha-smooth muscle actin staining, consistent with TIMP-induced phenotypic differentiation into myofibroblasts. Finally, infection with AdTIMP-2 resulted in a significant increase in collagen synthesis, whereas infection with AdTIMP-3 resulted in a significant increase in fibroblast apoptosis. TIMPs exert overlapping as well as diverse effects on isolated cardiac fibroblasts. The observation that TIMPs stimulate fibroblast proliferation as well as phenotypic differentiation into myofibroblasts suggests that TIMPs may play an important role in tissue repair in the heart that extends beyond their traditional role as MMP inhibitors.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Matrix metalloproteinases and their inhibitors,modulators of neuro-immune interactions and of pathophysiological processes in the nervous system

The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), that regulate or degrade by proteolytic cleavage protein components of the extracellular matrix, cytokines, chemokines, cell adhesion molecules and a variety of membrane receptors. MMP activity is counterbalanced by their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), a family of 4 secreted multifunctional proteins that have growth promoting activities. In physiological conditions MMP activity is tightly regulated and altered MMP regulation is associated with pathological processes including inflammation, cell proliferation, cell death and tissue remodeling. The MMP/TIMP system is involved in the development and function of cells of the immune system by promoting their differentiation, activation, migration across basement membranes and tissues. In the last years, data has accumulated indicating that the MMP/TIMP system is expressed in the nervous system where it regulates neuro-immune interactions and plays a major role in pathophysiological processes. In this review, we present recent in vivo and in vitro studies that highlight the contribution of the MMP/TIMP system to various diseases of the nervous system, involving blood brain barrier breakdown, neuroinflammation, glial reactivity, neuronal death, reactive plasticity, and to developmental and physiological processes including cell migration, axonal sprouting and neuronal plasticity. This review also alludes to the beneficial effects of synthetic MMP inhibitors in different animal models of neuropathology. In all, a further understanding of the role of MMPs and TIMPs in the nervous system should contribute to unravel mechanisms of neuronal plasticity and pathology and set the basis of new therapeutic strategies in nervous system disorders based on the development of synthetic MMP inhibitors.     

Crystal structures of MMPs in complex with physiological and pharmacological inhibitors

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.     

Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate

The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.