Matrix Metalloproteinases (MMPs)

• Metallo, serine, cysteine, aspartic acid, and threonine proteases are mechanistic classes of catalysis that selectively process, cleave, or degrade proteins.
• Proteases are fundamental enzymes that represent 3% of the human genome.

MMP Overview

• The first matrix metalloproteinase (MMP) was discovered in 1962, capable of degrading collagen.
• MMPs remodel the extracellular matrix and have roles in cell signaling, immune regulation, and transcriptional control.
• Initially overlooked, MMP contributions are both beneficial and detrimental in disease progression and resolution.
• Thousands of MMP substrates have been suggested, however, a few hundred have been validated.
• Identified enigmas need to be solved regarding their biological functions in diseases after 60 years of research.
• Focus is maintained on matrix metalloproteinases (MMPs) and examination of biochemical tools is performed to study their functions.

History of MMPs

• J. Frederick Woessner published a landmark paper in 1962 showing that a protease could degrade collagen in the mammalian uterus.
• Jerome Gross and Charles Lapiere described an MMP by demonstrating that the anuran tadpole underwent collagen turnover during metamorphosis in 1962.
• In 1966, the first MMP (MMP1), termed tadpole collagenase, was purified from a tadpole tail fin and back skin.
• MMP2 and MMP3 were identified in the 1970s.
• MMP2 was first sequenced in 1988 and was first called 72-kDa type IV collagenase/gelatinase A due to its ability to degrade denatured collagen/gelatin.
• Salvesen and Nagase first purified MMP2 from human rheumatoid synovial fibroblasts in 1990.
• MMP3, originally termed proteoglycanase, was identified due to its lower molecular mass (54 kDa) and its ability to degrade proteoglycan and casein.
• Werb first purified MMP3 from rabbit synovial fibroblasts in 1985 and later called it stromelysin.
• The first evidence of the cross-activation of MMPs in a protease web was in 1987 by the activation of proMMP1 (latent MMP1) by MMP3.
• MMP4, -5, and -6 were believed to be novel MMPs but were later found to be redundantly identified as MMP1, -2, or -3.
• The name MMP was first proposed in the late 1980s, and the International Union of Biochemistry and Molecular Biology designated the family with the terminology.
• There were seven known MMPs (MMP1, -2, -3, -7, -8, -9, and -10) by 1991.
• The MMP family in humans consists of 23 members, each composed of various domains.

MMP Domains, Structures, and Mechanism of Action for Proteolysis

• MMPs belong to the superfamily of metzincin proteases, which can be grouped into six families
• The metzincins received this denomination due to a characteristic feature in their structure, a methionine (Met) residue at the active site and the use of zinc in the enzymatic reaction.
• They share the following conserved domain: HEXXHXXGXXH, where H is histidine, E is glutamic acid, G is glycine, and X is any amino acid.
• The deep cavity's composition can be identical among its members, reaching up to 86% of similarity for MMP3 and -10.
• MMPs can be divided into subgroups depending on their linear sequence similarity, domain organization, and substrate specificity. For example, MMP1, -3, -8, -10, -12, -13, -19, -20, and -27 are archetypal MMPs containing a signal peptide, a prodomain, a catalytic domain, and a hemopexin-like C-terminal domain.
• MMP2 and -9 were previously called gelatinases A and B due to their ability to cleave denatured collagen or gelatin; they contain a fibronectin type-II modules domain.
• MMP7 and -26, initially termed matrilysins, lack a hemopexin-like domain.
• There are six membrane-type matrix metalloproteinases, or MT-MMPs that localize to the plasma membrane via a transmembrane segment or a glycosylphosphatidylinositol (GPI) anchor domain.
• MMP23 is a unique MMP that contains a cysteine array (CA) and immunoglobulin (Ig)-like domains, and the precise roles of these unique domains remain elusive.
• MMP28 or epilysin is the last member of the MMPs to be identified with features not observed in other MMPs.
• MMP1, -8, -13, and MT1-MMP can cleave fibrillar collagen types I to IV, and also XI, resulting in the generation of two typical fragments: 1/4 C-terminal and 3/4 N-terminal.
• MMP2 and -9 can also cleave extracellular matrix (ECM) proteins in vitro, including collagens and proteoglycans, resulting in changes in embryonic growth and development, tissue remodeling, inflammation, and wound healing.
• MMP7 and -26 can also cleave collagen and gelatin in vitro.
• MMP3 and -10 share the same domain presented by the archetypal MMPs but cannot cleave interstitial collagen.
• MMP3 and -10 cleave substrates and promote the conversion of a proMMP into an active enzyme.
• The MT-MMPs have a prodomain and are activated intracellularly via proteolytic processing of the furin domain, promoting activation of the enzyme that is further directed to the cell surface due to its membrane-anchoring domains.
• MMP28 mRNA is expressed in epithelial cells of many tissues in both mice and humans and appears to contain limited ability to cleave ECM proteins but has been shown to stimulate chemokine expression.
• MMP transcription is independently regulated, as each cell type may produce different MMPs and at different levels.
• functional AP-1 site was demonstrated to mediate MMP2 transcription in cardiac cells
• Changes in cell signaling via phosphorylation of mitogen-activated protein kinase (MAPK), p38, focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK)1, or protein kinase B (Akt) can induce MMP transcription and translation Lastly, there is a total of six binding pockets in MMPs: S1, S2, S3, S1', S2', and S3'
• MMPs are activated, need to be regulated by endogenous inhibitors.

Endogenous Inhibitors and the Tissue Inhibitor of Metalloproteinases

• First tissue inhibitor of metalloproteinase (TIMP) was described in 1972 (Bauer et al., 1972).
• TIMP1 was found to be identical to a factor with erythroid-potentiating activity (Gasson et al., 1985).
• TIMP2 was cloned and sequenced from an A2058 human melanoma cell cDNA library (Stetler-Stevenson et al., 1990).
• TIMP2 was demonstrated to inhibit collagenase activity supporting the role of TIMPs. Four TIMPs are variably glycosylated and have molecular masses that exhibit basic similarities.
• the first is an approximately 125 amino acid N-terminal domain, and the second is an approximately 65 amino acid C-terminal domain
• The N-terminal domain is critical for inhibiting metalloproteinase activity.
• the C-terminal domain of TIMPs was thought to have a limited role in metalloproteinase inhibition
• TIMP2, TIMP3, and TIMP4 can interact with proMMP2, whereas TIMP1 and TIMP3 can interact with proMMP9 .
• Many tissues constitutively express the TIMP family and of TIMPs can be induced or inhibited -TIMP1 RNA is generally elevated in several inflammation. levels altered in response that are related to damage TIMP4 is the least studied. tissue and settings can be upregulated

Timps function through both dependent and metalloproteinase-independent mechanisms. TIMPs can all inhibit the active forms with lower binding constants.

• TIMP1 is also a poor inhibitor of MMP19.

• TIMP3 has a broad inhibition profile compared with the other TIMPs.

• ADAM10's activity was blocked by TIMP1 and -3 but not TIMP2 and -4.

• TIMP3 was also shown to suppresses MAPKs through TGFa in specific models