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.     

Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1

The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 A resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between beta-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.     

Domain specific overexpression of TIMP-2 and TIMP-3 reveals MMP-independent functions of TIMPs during Xenopus laevis development

Extracellular matrix remodelling mediates many processes including cell migration and differentiation and is regulated through the enzymatic action of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). TIMPs are secreted proteins, consisting of structurally and functionally distinct N- and C-terminal domains. TIMP N-terminal domains inhibit MMP activity, whereas their C-terminal domains may have cell signalling activity. The in vivo role of TIMP N- and C-terminal domains in regulating developmental events has not previously been demonstrated. Here we investigated the roles of TIMP-2 and TIMP-3 N- and C-terminal domains in Xenopus laevis embryos. We show that overexpression of TIMP-2 N- and C-terminal domains results in severe developmental defects and death, as well as unique changes in MMP-2 and -9 expression, indicating that the individual domains may regulate MMPs through distinct mechanisms. In contrast, we show that only the N-terminal, but not the C-terminal domain of TIMP-3, results in developmental defects.     

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.     

Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases.

The tissue inhibitors of matrix metalloproteinases (MMPs), TIMP-1 and TIMP-2, are also angiogenesis inhibitors. Cathepsin B and MMPs are found at sites of neovascularization in pathologies such as cancer and osteoarthritis. Treatment of TIMP-1, TIMP-2, and of a mixture of both inhibitors from human articular chondrocytes with cathepsin B resulted in their fragmentation, whereby they lost their MMP-inhibitory and anti-angiogenic activities. Our data suggest that, besides directly participating in tissue destruction, cathepsin B can be harmful for two further reasons: it raises the activity of the MMPs also in the absence of mechanisms up-regulating these enzymes, and it stimulates angiogenesis. This is a prerequisite for blood vessel invasion in a variety of pathological situations of which cancer and osteoarthritis are prominent examples.     

Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.     

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.     

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.     

Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis.

The matrix metalloproteinases (MMPs) constitute a multigene family of over 23 secreted and cell-surface associated enzymes that cleave or degrade various pericellular substrates. In addition to virtually all extracellular matrix (ECM) compounds, their targets include other proteinases, chemotactic molecules, latent growth factors, growth factor-binding proteins and cell surface molecules. The MMP activity is controlled by the physiological tissue inhibitors of MMPs (TIMPs). There is much evidence that MMPs and their inhibitors play a key role during extracellular remodeling in physiological situations and in cancer progression. They have other functions that promoting tumor invasion. Indeed, they regulate early stages of tumor progression such as tumor growth and angiogenesis. Membrane type MMPs (MT-MMPs) constitute a new subset of cell surface-associated MMPs. The present review will focus on MT1-MMP which plays a major role at least, in the ECM remodeling, directly by degrading several of its components, and indirectly by activating pro-MMP2. As our knowledge on the field of MT1-MMP biology has grown, the unforeseen complexities of this enzyme and its interaction with its inhibitor TIMP-2 have emerged, often revealing unexpected mechanisms of action.     

Matrix metalloproteinase-9,-3 and tissue inhibitor of matrix metalloproteinase-1 in colorectal cancer: relationship to clinicopathological variables

The balance between matrix metalloproteinases (MMPs) and their physiological tissue inhibitors of matrix metalloproteinases (TIMPs) is crucial in tumour invasion and progression. The aim of this study was to investigate the levels of MMP-9, MMP-3 and TIMP-1 in colorectal cancer (CRC) and to evaluate these proteinases and their inhibitor with respect to clinicopathological variables. Activities of pro- and active MMP-9 were measured in paired tumour and distant normal tissue specimens from 43 patients with CRC using gelatin zymography. ELISA was employed for the determination of MMP-9, MMP-3 and TIMP-1 protein expressions. The activity levels of pro- and active MMP-9 and protein expression levels of MMP-9, MMP-3 and TIMP-1 were higher in tumour tissues than in the corresponding normal tissues; the differences being significant for all (p < 0.05), except TIMP-1. Similarly, active MMP-9/proMMP-9 and the ratio of protein expression level of MMP-9-TIMP-1 were found to be significantly higher in tumour tissues ( p < 0.01). Among all the clinicopathological variables investigated, significant correlations were found between MMP-9 and presence of perineural invasion, MMP-3 and lymph node status, TIMP-1 and tumour differentiation, MMP-9/TIMP-1 ratio and histological types ( p < 0.05). In conclusion, MMP-3 was not as notably increased as MMP-9 in tumour tissues. However, different roles may be attributed to MMP-9 and MMP-3 in CRC development and progression. Additionally, assessment of TIMP-1 in relation to MMPs appeared to be crucial in CRC studies to provide a basis for the re-evaluation of the clinical usefulness of TIMP-1 in colorectal cancer.     

Expression level and glycan dynamics determine the net effects of TIMP-1 on cancer progression

Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer. [BMB Reports 2012; 45(11): 623-628]     

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.     

Effects of ovariectomy on aggregation,secretion, and metalloproteinases in porcine platelets

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.     

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.     

Inactivation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by Porphyromonas gingivalis

In response to periodontal inflammation, host cells release matrix metalloproteinases (MMPs) that contribute to periodontal tissue breakdown unless the tissue inhibitors of metalloproteinases (TIMPs) neutralize their activity. In this study, the capacity of Porphyromonas gingivalis to inactivate TIMP-1 was investigated. Proteolytic digestion of TIMP-1 was monitored by SDS-PAGE and Western immunoblotting. Planktonic cells and biofilms of P. gingivalis degraded TIMP-1 with production of several lower molecular mass fragments. Incorporation of human serum in the assay mixture had no effect on the degradation of TIMP-1 by P. gingivalis, whereas a cysteine proteinase inhibitor caused a complete inhibition. Using a fluorogenic assay, it was found that TIMP-1 treated with P. gingivalis lost its capacity to inhibit MMP-9 activity. This study revealed the potential of P. gingivalis to inactivate TIMP-1 through proteolytic degradation. This phenomenon may contribute to increasing significantly the level of active MMPs in affected periodontal sites and subsequently favor tissue destruction.     

TIMP-1 promotes VEGF-induced neovascularization in the retina

Proteolysis of vascular basement membranes and surrounding extracellular matrix is a critical early step in neovascularization. It requires alteration of the balance between matrix metalloproteinases (MMPs) and proteins that bind to and inactivate MMPs, tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 has been demonstrated to inhibit neovascularization in chick chorioallantoic membranes. However, TIMP-1 has also been shown to either promote or inhibit cell proliferation and migration in different settings. To determine whether genetic alteration of the MMP/TIMP-1 ratio would alter retinal neovascularization, we crossed mice that express vascular endothelial growth factor (VEGF) in photoreceptors with TIMP-1-deficient mice or mice that overexpress TIMP-1. Compared to VEGF transgene-positive/TIMP-1-sufficient mice, VEGF transgene-positive/TIMP-1-deficient mice showed smaller neovascular lesions. There was also no difference between the two groups of mice in the appearance of the neovascularization by light or electron microscopy. Compound VEGF/TIMP-1 transgenic mice had increased expression of both VEGF and TIMP-1 in the retina, and had more neovascularization than mice that had increased expression of VEGF alone. These gain- and loss-of-function data suggest that alteration of the TIMP-1/MMP ratio modulates retinal neovascularization in a complex manner and not simply by altering the proteolytic activity and thereby invasiveness of endothelial cells.     

Inhibition of matrix metalloproteinase activity in DU145 human prostate cancer cells by flavonoids from lowbush blueberry (Vaccinium angustifolium): possible roles for protein kinase C and mitogen-activated protein-kinase-mediated events

Regulation of the matrix metalloproteinases (MMPs) is crucial to regulate extracellular matrix (ECM) proteolysis which is important in metastasis. This study investigated the mechanism(s) by which three flavonoid-enriched fractions from lowbush blueberry (Vaccinium angustifolium) down-regulate MMP activity in DU145 human prostate cancer cells. Metalloproteinase activity was evaluated from cells exposed to "crude," anthocyanin-enriched (AN) and proanthocyanidin-enriched (PAC) fractions. Differential down-regulation of MMPs was observed. The activity of the endogenous tissue inhibitors of metalloproteinases (TIMPs) from these cells was also evaluated. Increases in TIMP-1 and TIMP-2 activity were observed in response to these fractions. The possible involvement of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways in the flavonoid-mediated decreases in MMP activity was observed. These findings indicate that blueberry flavonoids may use multiple mechanisms in down-regulating MMP activity in these cells.     

MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors

The goal of our study was to analyse the prognostic values for some matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in breast cancer. We evaluated the activity and the expression levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in malignant versus benign fresh breast tumor extracts. For this purpose, gelatinzymography, immunoblotting and ELISA were used to analyse the activity and expression of MMPs and TIMPs. We found that MMP-9 expression level and activity are increased in malignant tumors. In addition, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio values obtained by us were significantly different in malignant tumors compared to benign tumors. We suggest that the abnormal MMP-9/TIMP-1 balance plays a role in the configuration of breast invasive carcinoma of no special type and also in tumor growth, while altered MMP-2/TIMP-2 ratio value could be associated with lymph node invasion and used as a prognostic marker in correlation with Nottingham Prognostic Index. Finally, we showed that in malignant tumors high expression of estrogen receptors is associated with enhanced activity of MMP-2 and increased bcl- 2 levels, while high expression of progesterone receptors is correlated with low TIMP-1 protein levels.