BBA - Molecular Cell Research (v.1803, #1)

Matrix Metalloproteinases by Rafael Fridman (1-2).

Matrix metalloproteinases: Evolution, gene regulation and functional analysis in mouse models by Miriam Fanjul-Fernández; Alicia R. Folgueras; Sandra Cabrera; Carlos López-Otín (3-19).
Matrix metalloproteinases (MMPs) are a large family of zinc-endopeptidases which play important roles in multiple physiological and pathological processes. These enzymes are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplication, gene fusion and exon shuffling events to generate the multidomain architecture and functional diversity currently exhibited by MMPs. Proper regulation of these enzymes is required to prevent their unwanted activity in a variety of disorders, including cancer, arthritis and cardiovascular diseases. Multiple hormones, cytokines and growth factors are able to induce MMP expression, although the tissue specificity of the diverse family members is mainly achieved by the combination of different transcriptional control mechanisms. The integration of multiple signaling pathways, coupled with the cooperation between several cis-regulatory elements found at the MMP promoters facilitates the strict spatiotemporal control of MMP transcriptional activity. Additionally, epigenetic mechanisms, such as DNA methylation or histone acetylation, may also contribute to MMP regulation. Likewise, post-transcriptional regulatory processes including mRNA stability, protein translational efficiency, and microRNA-based mechanisms have been recently described as modulators of MMP gene expression. Parallel studies have led to the identification of MMP polymorphisms and mutations causally implicated in the development of different genetic diseases. These genomic analyses have been further extended through the generation of animal models of gain- or loss-of-function for MMPs which have allowed the identification of novel functions for these enzymes and the establishment of causal relationships between MMP dysregulation and development of different human diseases. Further genomic studies of MMPs, including functional analysis of gene regulation and generation of novel animal models will help to answer the multiple questions still open in relation to a family of enzymes which strongly influence multiple events in life and disease.
Keywords: Degradome; Protease; Cancer; Polymorphism; Cardiovascular disease;

Matrix metalloproteinases: Fold and function of their catalytic domains by Cynthia Tallant; Aniebrys Marrero; F.Xavier Gomis-Rüth (20-28).
Matrix metalloproteinases (MMPs) are zinc-dependent protein and peptide hydrolases. They have been almost exclusively studied in vertebrates and 23 paralogs are present in humans. They are widely involved in metabolism regulation through both extensive protein degradation and selective peptide-bond hydrolysis. If MMPs are not subjected to exquisite spatial and temporal control, they become destructive, which can lead to pathologies such as arthritis, inflammation, and cancer. The main therapeutic strategy to combat the dysregulation of MMPs is the design of drugs to target their catalytic domains, for which purpose detailed structural knowledge is essential. The catalytic domains of 13 MMPs have been structurally analyzed so far and they belong to the “metzincin” clan of metalloendopeptidases. These compact, spherical, ∼ 165-residue molecules are divided by a shallow substrate-binding crevice into an upper and a lower sub-domain. The molecules have an extended zinc-binding motif, HEXXHXXGXXH, which contains three zinc-binding histidines and a glutamate that acts as a general base/acid during catalysis. In addition, a conserved methionine lying within a “Met-turn” provides a hydrophobic base for the zinc-binding site. Further earmarks of MMPs are three α-helices and a five-stranded β-sheet, as well as at least two calcium sites and a second zinc site with structural functions. Most MMPs are secreted as inactive zymogens with an N-terminal ∼ 80-residue pro-domain, which folds into a three-helix globular domain and inhibits the catalytic zinc through a cysteine imbedded in a conserved motif, PRCGXPD. Removal of the pro-domain enables access of a catalytic solvent molecule and substrate molecules to the active-site cleft, which harbors a hydrophobic S1′-pocket as main determinant of specificity. Together with the catalytic zinc ion, this pocket has been targeted since the onset of drug development against MMPs. However, the inability of first- and second-generation inhibitors to distinguish between different MMPs led to failures in clinical trials. More recent approaches have produced highly specific inhibitors to tackle selected MMPs, thus anticipating the development of more successful drugs in the near future. Further strategies should include the detailed structural characterization of the remaining ten MMPs to assist in achieving higher drug selectivity. In this review, we discuss the general architecture of MMP catalytic domains and its implication in function, zymogenic activation, and drug design.
Keywords: Zinc metalloproteinase; Metzincin; MMP; Vertebrate collagenase; Three-dimensional structure; Catalytic domain; X-ray crystal structure; Metallopeptidase;

The zinc-dependent matrix metalloproteinases (MMPs) belong to a large family of structurally homologous enzymes. These enzymes are involved in a wide variety of biological processes ranging from physiological cell proliferation and differentiation to pathological states associated with tumor metastasis, inflammation, tissue degeneration, and cell death. Controlling the enzymatic activity of specific individual MMPs by antagonist molecules is highly desirable, first, for studying their individual roles, and second as potential therapeutic agents. However, blocking the enzymatic activity with synthetic small inhibitors appears to be an extremely difficult task. Thus, this is an unmet need presumably due to the high structural homology between MMP catalytic domains. Recent reports have recognized a potential role for exosite or allosteric protein regions, distinct from the extended catalytic pocket, in mediating MMP activation and substrate hydrolysis. This raises the possibility that MMP enzymatic and non-enzymatic activities may be modified via antagonist molecules targeted to such allosteric sites or to alternative enzyme domains. In this review, we discuss the structural and functional bases for potential allosteric control of MMPs and highlight potential alternative enzyme domains as targets for designing highly selective MMP inhibitors.
Keywords: Allostery; Allosteric regulation; Matrix metalloproteinase (MMP); Protein flexibility/hemopexin domain; Fibronectin domain; Exosite; Protein conformation;

The biological roles of the matrix metalloproteinases (MMPs) have been traditionally associated with the degradation and turnover of most of the components of the extracellular matrix (ECM). This functional misconception has been used for years to explain the involvement of the MMP family in developmental processes, cell homeostasis and disease, and led to clinical trials of MMP inhibitors for the treatment of cancer that failed to meet their endpoints and cast a shadow on MMPs as druggable targets. Accumulated evidence from a great variety of post-trial MMP degradomics studies, ranging from transgenic models to recent state-of-the-art proteomics screens, is changing the dogma about MMP functions. MMPs regulate cell behavior through finely tuned and tightly controlled proteolytic processing of a large variety of signaling molecules that can also have beneficial effects in disease resolution. Moreover, net proteolytic activity relies upon direct interactions between the different protease and protease inhibitor families, interconnected in a complex protease web, with MMPs acting as key nodal components. Such complexity renders simple interpretation of Mmp knockout mice very difficult. Indeed, the phenotype of these models reveals the response of a complex system to the loss of one protease rather than necessarily a direct effect of the lack of functional activity of a protease. Such a shift in the MMP functional paradigm, together with the difficulties associated with current methods of studying proteases this highlights the need for new high content degradomics approaches to uncover and annotate MMP activities in vivo and identify novel interactions within the protease web. Integration of these techniques with specifically designed animal models for final validation should lay the foundations for the development of new inhibitors that specifically target disease-related MMPs and/or their upstream effectors that cause deleterious effects in disease, while sparing MMP functions that are protective.
Keywords: MMP; Protease; Degradomics; Protease web; Proteomics; Mass spectrometry;

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.
Keywords: Matrix metalloproteinase; Extracellular matrix; Evolution; Multifunctional protein; Cell growth; Sorsby fundus dystrophy;

To bind zinc or not to bind zinc: An examination of innovative approaches to improved metalloproteinase inhibition by Jennifer A. Jacobsen; Jody L. Major Jourden; Melissa T. Miller; Seth M. Cohen (72-94).
This short review highlights some recent advances in matrix metalloproteinase inhibitor (MMPi) design and development. Three distinct approaches to improved MMP inhibition are discussed: (1) the identification and investigation of novel zinc-binding groups (ZBGs), (2) the study of non-zinc-binding MMPi, and (3) mechanism-based MMPi that form covalent adducts with the protein. Each of these strategies is discussed and their respective advantages and remaining challenges are highlighted. The studies discussed here bode well for the development of ever more selective, potent, and well-tolerated MMPi for treating several important disease pathologies.
Keywords: Zinc-binding group; Mechanism-based inhibitor; MMP;

Avoiding spam in the proteolytic internet: Future strategies for anti-metastatic MMP inhibition by Achim Krüger; Ronald E. Kates; Dylan R. Edwards (95-102).
Phase III clinical trials with cancer patients with the first generation of synthetic MMP inhibitors (MMPIs) failed due to inefficacy and adverse side effects. These results were unexpected, given the wealth of pre-clinical data implicating MMPs as cancer targets, but are attributable to the broad-spectrum activity of these early MMPIs and the limited knowledge of the variety of biological functions of MMPs at the time they were deployed. These experiences stimulated the development of a variety of highly specific synthetic MMPIs. However, the bottle-neck is the identification of true target-MMPs. Functional genetic approaches are being complicated by the existence of the ‘protease web,’ i.e., the dynamic interconnectivity of MMPs and other proteases, their inhibitors, and substrates that collectively establish homeostasis in signaling in healthy and disease-afflicted tissue. Therefore, even specific MMP inhibition can result in seemingly unpredictable induction of systemic protease web-associated modulations (spam), which can comprise metastasis-promoting molecules such as other proteases and cytokines. Such undesired information in local proteolytic networks or relayed systemically in the organism via the proteolytic internet needs to be understood and defined in order to design specific metastasis therapies employing highly specific MMPIs in combination with spam-filtering agents.
Keywords: MMP inhibitor; Protease web; Proteolytic internet; Metastasis;

A number of extensive reviews are available discussing the roles of MMPs in various aspects of cancer progression from benign tumor formation to overt cancer present with deadly metastases. This review will focus specifically on the evidence functionally linking the MMPs and tumor-induced angiogenesis in various in vivo models. Emphasis has been placed on the cellular origin of the MMPs in tumor tissue, the requirement of proMMP activation and the resulting proteolytic activity for the induction and progression of tumor angiogenesis, and the pleiotropic roles for some of the MMPs. The functional mechanisms of the angiogenic MMPs are discussed as well as their catalytic detection in complex biological systems. In addition, the contribution of active MMPs to metastatic spread and establishment of secondary metastasis will be discussed in view of the findings indicating that MMPs are involved in the preparation of pre-metastatic niches. Finally, the most recent evidence, indicating the pro-metastatic consequences of anti-angiogenic therapies employing MMP inhibitors will be presented as examples highlighting possible outcomes of interfering with the pleiotropic nature of the MMP functionality.
Keywords: Cancer; Metastasis; Matrix metalloproteinases; Tumor angiogenesis; Angiogenic switch; VEGF; FGF-2; Neutrophil; MMP-9;

The metalloproteinase family includes MMP, ADAM and ADAMTS proteases. Mice deficient in individual or pairs of metalloproteinases have been generated, and a number of these genetic models spontaneously develop skeletal abnormalities. Here we review metalloproteinase function in endochondral and intramembranous ossification, as well as in postnatal bone remodeling. We highlight how metalloproteinases enable interactions between distinct bone cell types and how this communication contributes to the skeletal phenotypes observed in knockout mice. In addition to the physiological actions of metalloproteinases in the skeletal system, the experimental manipulation of metalloproteinase-deficient mice has revealed substantial roles for these enzymes in osteoarthritis and rheumatoid arthritis. MMP, ADAM and ADAMTS proteases thus emerge as key players in the development and homeostasis of the skeletal system.
Keywords: Metalloproteinase; Skeletal development; Arthritis; Ossification; Genetically engineered mouse model;

This manuscript provides an overview of the dynamic interactions which play an important role in regulating cancer cell functions. We describe and discuss, primarily, those interactions which involve membrane type-1 matrix metalloproteinase (MT1-MMP), its physiological inhibitor tissue inhibitor of metalloproteinases-2 (TIMP-2), furin-like proprotein convertases and the low density lipoprotein-related protein 1 (LRP1) signaling scavenger receptor. The interaction among these cellular proteins controls the efficiency of the activation of MT1-MMP and the unorthodox intracellular signaling which is generated by the catalytically inert complex of MT1-MMP with TIMP-2 and which plays a potentially important role in the migration of cancer cells. Our in-depth understanding of these cellular mechanisms may provide the key to solving the puzzling TIMP-2 paradox. This unsolved paradox arises from the fact that TIMP-2 is a powerful inhibitor of MMPs including MT1-MMP, but at the same time high levels of TIMP-2 positively correlate with an unfavorable prognosis in cancer patients. Solving the TIMP-2 paradox may lead to solving a similar PAI-1 paradox and produce a clearer understanding of the biochemical mechanisms which control the functionality of the urokinase-type plasminogen activator•urokinase receptor•plasminogen activator inhibitor type-1 (uPAR•uPA•PAI-1) system in cancer.
Keywords: Matrix metalloproteinase; Furin; LRP1; TIMP-2; Cell migration; Metastasis;

Pericellular proteolysis mediated by membrane-type 1 matrix metalloproteinase (MT1-MMP) represents an essential component of the cellular machinery involved in the dissolution and penetration of ECM barriers by tumor cells. Although most studies on the proinvasive properties of MT1-MMP have focused on its unusually broad proteolytic activity towards several ECM components and cell surface receptors, recent evidence indicate that the cytoplasmic domain of the enzyme also actively participates in tumor cell invasion by regulating the cell surface localization of MT1-MMP as well as the activation of signal transduction cascades. The identification of the molecular events by which the intracellular domain of MT1-MMP links proteolysis of the surrounding matrix by the enzyme to modification of cell function may thus provide important new information on the mechanisms by which this enzyme controls the invasive behavior of neoplastic cells in vivo.
Keywords: MT1-MMP; Cytoplasmic domain; Signal transduction;