Structural basis of the matrix metalloproteinases and their physiological inhibitors,the tissue inhibitors of metalloproteinases

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.     

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.     

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.     

基质金属蛋白酶

基质金属蛋白酶是一类分解细胞外基质组分的锌蛋白酶⒚它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用⒚基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节⒚基质金属蛋白酶以酶原形式分泌,随后被其它蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活⒚所有基质金属蛋白酶都受到天然抑制剂 金属蛋白酶组织抑制剂所抑制⒚两者的不平衡导致许多疾病的发生,如肿瘤侵入及转移⒚合成基质金属蛋白酶组织抑制剂所抑制,如 Ｍ ａｒｉｍ ａｓｔａｔ 能控制肿瘤转移的发生及进一步扩散⒚本文将对基质金属蛋白酶的特征、分子区域结构、底物特性、激活机制、调控方式等方面进行最新概述⒚     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mRNA expression during rat central nervous system development

The elucidation of the cellular and molecular mechanisms governing the maturation of the central nervous system (CNS) is rapidly emerging. Cell-cell and cell-matrix interactions play critical roles in all phases of developmental tissue remodeling. Throughout development, an intricate balance between extracellular matrix synthesis and degradation is preserved by the opposing actions of matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Although recent evidence suggests that TIMPs exert diverse cell biological functions distinct from their MMP-inhibitory activities, few studies have investigated MMP or TIMP expression during CNS development. The present report analyzes the mRNA expression of the four known TIMPs throughout the course of embryonic and postnatal rat CNS development. The results clearly demonstrate the unique spatial distribution and temporal regulation of TIMP expression and suggest a distinct role for each TIMP during CNS development.     

Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell‐associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re‐evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial–mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold. J. Cell. Biochem. 118: 3531–3548, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Computational Sequence Analysis of the Tissue Inhibitor of Metalloproteinase Family

The tissue inhibitor of metalloproteinase (TIMP) family regulates extracellular matrix turnover and tissue remodeling by forming tight-binding inhibitory complexes with matrix metalloproteinases (MMPs). MMPs and TIMPs have been implicated in many normal and pathological processes, such as morphogenesis, development, angiogenesis, and cancer metastasis. This minireview provides information that would aid in classification of the TIMP family and in understanding the similarities and differences among TIMP members according to the physical data, primary structure, and homology values. Calculations of molecular weight, isoelectric point values, and molar extinction coefficients are reported. This study also compares sequence similarities and differences among the TIMP members through calculations of homology within their individual loop regions and the mature region of the molecule. Lastly, this report examines structure–function relationships of TIMPs. Thorough knowledge of TIMP primary and tertiary structure would facilitate the uncovering of the molecular mechanisms underlying metalloproteinase, inhibitory activities and biological functions of TIMPs.     

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.     

Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.     

Unripe Rubus coreanus Miquel suppresses migration and invasion of human prostate cancer cells by reducing matrix metalloproteinase expression

Rubus coreanus Miquel (RCM) is used to promote prostate health and has been shown to have anti-oxidant and anti-carcinogenic activities. However, the effects and mechanisms of RCM on prostate cancer metastasis remain unclear. PC-3 and DU 145 cells were treated with ethanol or water extract of unripe or ripe RCM and examined for cell invasion, migration, and matrix metalloproteinases (MMPs) activity and expression. Phosphoinositide 3-kinase (PI3K) and Akt activities were examined. Unripe RCM extracts exerted significant inhibitory effects on cell migration, invasion, and MMPs activities. A significant reduction in MMPs activities by unripe RCM ethanol extract treatment (UE) was associated with reduction of MMPs expression and induction of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, PI3K/Akt activity was diminished by UE treatment. In this study, we demonstrated that UE decreased metastatic potential of prostate cancer cells by reducing MMPs expression through the suppression of PI3K/Akt phosphorylation, thereby decreasing MMP activity and enhancing TIMPs expression.     

Ligation induced matrix-metalloproteinase-9 activity in peripheral frog nervous system

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). MMP-2 and MMP-9 are two of the MMPs which are essential to contribute to inflammatory and degenerative processes in injured nerves. The aim of the present study was to examine expression and activities of MMP-2 and MMP-9 in the injured and control groups frog sciatic nerves using gelatin zymography. Our investigation demonstrated for the first time as far as we know the expression of MMP-2 and MMP-9 in frog sciatic nerve. The expression and activity of MMP-9 were increased two fold on average following ligation. By contrast, MMP-2 activities remained unchanged. These findings suggest that we can consider MMP-9 as a marker for degenerative changes that follow nerve ligation in frog nerve.     

Tissue inhibitors of metalloproteinases and programmed cell death: conundrums, controversies and potential implications

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which can synergistically degrade the major components of extracellular matrix (ECM). A key role in maintaining the balance between ECM deposition and degradation in several physio-pathological processes is carried out, through multiple biological functions, by four members of the tissue inhibitors of metalloproteinases (TIMPs) family. TIMP-1 and TIMP-2 are capable of inhibiting the activities of MMPs, can inhibit tumour growth, invasion and metastasis, exhibit growth factor-like activity, can inhibit angiogenesis and suppress programmed cell death (PCD) independently of the MMP-inhibitory activity. TIMP-3 is the only member which is tightly bound to ECM, inhibits TNF- converting enzyme and induces PCD through the stabilization of TNF- receptors on the cell surface. TIMP-4 plays a role in ECM homeostasis in a tissue-specific fashion and its overexpression induces PCD. The aim of this article is to review the exciting and intriguing literature on TIMPs, with special emphasis on their conflicting-paradoxical roles in PCD and their potential clinical usefulness.