Metalloproteinase or metalloprotease is a protease enzyme whose catalytic mechanism involves a metal (zinc or cobalt). It is the most diverse type of protease, with more than 50 families classified. The metal ion is interacted with the protein via three ligands which consist of histidine, glutamate, aspartate, lysine, and arginine. There is another binding site in the metalloproteinase that is absorbed by the unstable water molecules. Exopeptidases and endopeptidases, including ADAM proteins and matrix metalloproteinases, are two kinds of well-known metalloproteases. EDTA (metal chelator removing zinc) and orthophenanthroline are two main chelating agents that can lead to complete inactivated metalloproteases. Mounting evidence supports that metalloproteases play an important role in many physiological processes, including tumor tumorigenesis and tumor progression, chronic venous disease and muscle damage.
Structure of Metalloproteases
Around half of the known metalloproteases contain an HEXXH motif to form part of the metal-binding site. It can be more stringently defined for metalloproteases as 'abXHEbbHbc', where 'a' most often represents valine or threonine, 'b' is an uncharged residue, and 'c' is a hydrophobic residue. Proline can be adopted by this 'abXHEbbHbc' motif in metalloproteases and would break the helical structure, thus is never found in this site. Basic domain structure of MMPs includes the signal peptide domain for guiding the enzyme into the rough endoplasmic reticulum during protein synthesis, the propeptide domain for sustaining the latency of these enzymes until it is removed or disrupted, the catalytic domain, which houses the highly conserved Zn 2+ binding region and is responsible for enzyme activity, the hemopexin domain, which determines the substrate specificity of MMPs, and a small hinge region enabling the hemopexin region to present substrate to the active core of the catalytic domain. MMPs are classified into collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MTMMPs), and other MMPs according to their substrates and the organization of their different domains. Membrane-type MMPs (MT-MMPs) possess an additional intracellular domain and transmembrane domain. MT-MMPs also contain a cleavage site for furin proteases, providing the basis for furin-dependent activation of latent MT-MMPs before secretion. MMPs are produced in a latent form and most are activated by extracellular proteolytic cleavage of the propeptide. ADAMs is composed of propeptide, disintegrin-like, cysteine-rich, and epidermal growth factor–like domains. Especially, membrane-anchored ADAMs contain a transmembrane and cytoplasmic domain.
Regulation of MMP Activity
The function of MMPs in vivo is regulated by the balance between them and their physiological inhibitors. The proteolytic activity of MMPs can be regulated by many factors, including gene expression, compartmentalization, conversion from zymogen to active enzyme, and the presence of specific inhibitors. There are a variety of regulatory cascades that determine the functions of the diverse MMPs expressed in the complex tumor microenvironment. Many proteinases are overexpressed in tumor tissues and are synthesized as pre–pro-MMPs. Once activated, the signal peptide is removed to generate pro-MMPs. The cysteine from the “cysteine switch” motif coordinates with the catalytic Zn2+ to keep pro-MMP inactive. Then the cysteine switch is then often cleaved by other proteinases or MMPs to activate proMMPs. The tissue inhibitors of metalloproteinases (TIMPs) are the most important physiological inhibitors of MMP. It is also usually expressed at the tumor site. TIMP-1, -2, -3, and 4 form 1:1 isomeride complexes with active MMPs leading to inhibition of proteolytic activity. Similar to MMPs, the proteolytic ADAM and ADAMTS family members are inhibited by specific TIMPs. MMPs can also be activated by heat, N-ethylmaleimide, oxidized glutathione, sodium dodecyl sulfate, low pH, thiol-modifying agents such as 4-aminophenylmercuric acetate, mercury chloride, and chaotropic agents by interfering with the cysteine-Zn21 coordination at the cysteine switch. In addition, there are also specific MMP activators, including plasmin for MMP-9 and MMP-3, hypochlorous acid for MMP-7, and MMP-7 for MMP-1.
Figure 1. Proteolytic Cascades Regulate MMP Function
Metalloproteases Regulated Signaling Pathway
MMPs play a major role in lots of physiological processes by manipulating ECM proteins. ECM provides support and anchorage for cells, segregates tissues from one another, regulates cell movement and intercellular communication, and provides a local depot for cellular growth factors. It is mainly composed of fibers, proteoglycans, and polysaccharides. Fibers mainly include collagen (the main extracellular protein), and elastin (providing flexibility for the skin, arteries, and lungs). Proteoglycans act to attract water to keep the ECM hydrated, bind and store growth factors, and thus contain more carbohydrates than proteins. ECM also contains other proteins, glycoproteins, polysaccharides, and proteolytic enzymes. Among these, proteolytic enzymes continuously cause renewal of ECM proteins, whose components provide the structural scaffolding for blood vessel support, cell migration, differentiation and signaling, as well as epithelialization and wound repair. Collagen and elastin are essential for structural integrity of the venous wall and are important MMP substrates. In addition, matrix metalloproteinases can act as regulators of the tumor microenvironment, periodontal inflammation, chronic venous disease and muscle damage. Therefore, targeted metalloproteinases have significant potency during disease treatment.
Figure. 2 MMPs contributing to disease progression in inflammation as well as cancer of lungs and the gut