Activation of Metalloproteinases

The final products of MMP gene translation are inactive forms of enzymes such as zymogens or proMMPs. Functionally, the active form of MMPs requires exposure of the active catalytic center, which occurs via a series of conformational changes.

The active site containing a zinc-binding motif and surrounding amino acids consists of a cleft along the catalytic domain. In the latent form of the enzyme, the polypeptide tail of the prodomain region covers the catalytically competent cleft. The inhibitory cysteine with its sulfhydryl group acts as a fourth ligand and thus blocks accessibility to the active zinc atom. Cleavage of the protease-sensitive region in the prodomain causes translocation of the polypeptide tail away from the cleft, which upon removal of the cysteine from the active region, exposes the active zinc in coordination with a water molecule. The removal of the inhibitory prodomain exposes the substrate binding region located close to the active site. When the substrate binds and comes close to the active site, the carbonyl group of the substrate peptide bond replaces the water molecule and coordinates the active zinc. The glutamic acid located within the MMP active domain and adjacent to active zinc acts as a proton acceptor from water and provokes a nucleophilic attack on the peptide bond of substrate [5].

MMP activating factors include proteolytic enzymes that digest a protease-sensitive region in the prodomain. Glutathione and other thiol-modifying agents in general, chaotropic agents, SDS, reactive oxygen, NO, heat, or low pH can also perturb the molecular interaction between the inhibitory cysteine and the active zinc, causing activation of MMPs. MMP activation is a multistep process, involving initially the partial removal of the peptide from the prodomain by any of the above listed agents, culminating with the full removal of activation peptide of intermediate molecules.

Membrane-type MMPs contain a specific sequence between the propeptide and the catalytic domain that is susceptible for furin or furin-like proprotein convertases

Table I Groups of Human MMPs.

Enzyme

GENE

Domains

Substrate

Collagenases MMP-1,

Interstitial collagenase, collagenase 1

Neutrophil collagenase, collagenase 2 MMP-13 Collagenase 3

Gelatinases MMP-2, Gelatinase A

11q22—q23 Pre-Pro(SH>Cat(Zn)-Hinge-Hemopexin(S-S)

11q21—q22 Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin(S-S)

11q22.3 Pre-Pr(SH)-Cat(Zn)-Hinge-Hemopexin(S-S)

MMP-9, Gelatinase B

Stromelysins MMP-3, Stromelysin 1

MMP-10, Stromelysin 2

MMP-11, Stromelysin 3

Matrilysins

Matrilysin 1, pump-1

MMP-26,

Matrilysin 2, endometase

Membrane-Type MMPs MMP-14, MT1-MMP

MMP-15, MT2-MMP

MMP-16, MT3-MMP

MMP-17, MT4-MMP

MMP-24, MT-MMP

MMP-25, MT6-MMP

16q13

11q23

11q22.3-q23 22q11.2

11q21—q22

11p15

14q11—q12

15q13—q21

8q21

12q24.3

20q11.2

16p13.3

Pre-Pro(SH)-Cat-3xFnII(Zn)-Hinge-Hemopexin(S-S)

Pre-Pro(SH)-Cat-3xFnII(Zn)-Hinge-Hemopexin(S-S)

Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin(S-S)

Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin(S-S)

Pre-Pro(SH)FurinSite-Cat(Zn)-Hinge-Hemopexin(S-S)

Pre-Pro(SH)FurinSite-Cat(Zn)-Hinge-Homopexin(S-S)-TM-Cytoplasmic tail

Pre-Pro(SH)FurinSite Homopexin(S-S)-TM-Pre-Pro(SH)FurinSite Homopexin(S-S)-TM-Pre-Pro(SH)FurinSite Homopexin(S-S)-GPI

Pre-Pro(SH)FurinSite Homopexin(S-S)-TM-

Pre-Pro(SH)FurinSite Homopexin(S-S)-GPI

Cat(Zn)-Hinge-Cytoplasmic tail Cat(Zn)-Hinge-Cytoplasmic tail

Cat(Zn)-Hinge-anchor

Cat(Zn)-Hinge-Cytoplasmic tail

Cat(Zn)-Hinge-anchor

Aggrecan, collagens I, II, III, VII, VIII, X, XI, enactin, fibronectin, gelatin, IGFBPs, laminin, link protein, myelin basic, tenascin, vitronectin, a1-AC, a2-M, a1-PI, casein, C1q, fibrin, fibrinogen, IL1 b, ProTNFa

Aggrecan, collagens I, II, III, a2-M, a1-PI, C1q, fibrinogen, substance P

Aggrecan, collagens I, II, III, VI, IX, X, XIV, fibrillin, fibronectin, gelatin 1, osteonectin, a2-M, casein, factor XII, fibrinogen, proMMP2, ProTNFa

ADP-ribose polymerase (in vitro) [ref 64 S1], collagen I, III, IV, V, VII, IX, X, XI, decorin, elastin, entactin/ nidogen, fibrillin, fibronectin, fibulins, gelatinl, IGFBPs, laminin, a1-AC, a1-PI, C1q, fibrin, fibrinogen, ILlß, ProTNFß, plasminogen substance P

Aggrecan, collagens IV, V, XI, XIV, decorin, elastin, fibrillin, gelatinl, laminin, link protein, myelin basic, osteonectin, vitronectin, a2-M, al-PI, casein, Clq, fibrin, fibrinogen, ILlß, ProTNFa, Pro TNFß, plasminogen, substance P

Aggrecan, collagens III, IV, V, VII, IX, X, XI, decorin, elastin, entactin/nidogen, fibrillin, elastin, fibronectin, gelatin1, IGFBPs, laminin, link protein, myelin basic, osteonectin, tenascin, vitronectin, al-AC, al-PI, Clq, E-cadherin, fibrin, fibrinogen, ILlß, ProTNFa, plasminogen,substance P, T-kininogen

Aggrecan, collagens III, IV, V, elastin, fibronectin, gelatinl, link protein, casein, fibrinogen

Aggrecan, collagens I, IV, decorin, elastin, entactin/ nidogen, fibronectin, fibulins, gelatin1, laminin, link protein, myelin basic, osteonectin, tenascin, vitronectin, a1-PI, casein, E-cadherin, fibrinogen, Pro-a-defensin, Fas-ligand, Pro(TNF)-a

Collagen IV, fibronectin, gelatin1, a1-PI, fibrinogen

Aggrecan, collagens I, II, III, elastin, entactin/nidogen, fibronectin, gelatin l, laminin, vitronectin, a2-M, al-PI, Factor XII, fibrin, fibrinogen, ProMMP2, ProTNFa

Collagen III, fibronectin, ProMMP2

Table I Continued

Enzyme

GENE

Domains

Substrate

Others MMP-12, macrophage metalloelastase

MMP-19, RASI-1

MMP-20, enamelysin

MMP-23, cysteine array MMP (CA-MMP) MMP-27, CMMP

MMP-28, epilysin

11q22.2-q22.3 Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin

12q14 Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin

11q22.3 Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin

1p36.3 Pre-Pro(SH)-FurinSite-Cat(Zn)-Cys-rich-

Ig-like

11q24 Pre-Pro(SH)-Cat(Zn)-Hinge-Hemopexin

17q21.1 Pre-Pro(SH)FurinSite-Cat(Zn)-Hinge-

Hemopexin(S-S)

Aggrecan, collagens I, IV, decorin, elastin, entactin/ nidogen, fibronectin, gelatin 1, laminin, myelin basic, tenascin, a2-M, a1-PI, Factor XII, fibrinogen ProTNFa, ProTNFb, plasminogen

Collagens I, IV, fibronectin, gelatin 1, tenascin, casein amelogenin

Enzymes MMP-22 from chicken, MMP-18 and MMP-21 from Xenopus have been omitted.

Abbreviations: Pre, preprodomain, signal peptide; Pro(SH), prodomain with a zinc-ligating thiol (SH) group; FurinSite, specific sequence susceptible for furin cleavage located within Prodomain; Cat(Zn), catalytic domain with active zinc; 3xFnII, three Fibronectin-like domains inserted into a catalytic domain; Hinge, hinge region, linker polypetide; Homopexin(S-S), hemopexin domain with four propeller domains and a disulfide bridge between the first and fourth domain; TM, transmembrane region of protein; C, cytoplasmic tail, intracellular region of MMp expressed on the cell surface; GPI, glyco-phophatidyl inositol anchoring domain; Cys-rich, cystein and proline rich domain; Ig-like, immunoglobulin like domain. This table is based on [2, 4].

cleavage. During post-translational processing, these enzymes recognize and cleave within this sequence, thus activating MT-MMPs. Once transported through the Golgi network, activated MT-MMPs remain as surface enzymes at the cell membrane. Internalization of the enzyme can occur within 60 minutes after initial cell membrane exposure. This might represent a rapid response for relocalizing active MT1-MMP at the leading edge of the cell during migration.

Active MMPs and MT-MMPs degrade several components of ECM molecules. Their targets include other pro-teinases, proteinase inhibitors, clotting factors, chemotactic molecules, latent growth factors, growth factor-binding proteins, cell surface receptors, and cell-cell adhesion molecules. In addition, they may act via cell receptors, as for instance a2b1 integrin, thus affecting cell survival. Hence, the mechanisms of in vivo activation and inhibition of proMMPs are of high physiological importance. It was suggested that activation may take place on the external cell surface, where plasma plasminogen, together with its uPA/uPAR complex, is converted into plasmin, and plasmin then removes part of the propeptide of a freshly secreted MMP zymogen. Membrane-type MMP also plays a role in MMP activation by cleaving the rest of the propeptide. There is some evidence that this process is mediated by a specific MMP inhibitor (TIMP) that has high affinity to both the MT-MMP and MMP zymogen, keeping the complex docked on the cell surface. The adjacent MT-MMP, which is not occupied by a TIMP, then partially cleaves MMP zymogen, which in turn can auto-activate to the fully active form. Recently, a new activating agent was revealed that offers an additional control of MMP function called the SIBLINGS

proteins. Also, it was found that blood proteins involved in coagulation/fibrinolysis cascade, such as Activated Protein C, can directly activate MMPs.

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