Lecture 13 - Matrix Metalloproteinases.docx

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Cell and Systems Biology
Maurice Ringuette

Lecture 13 - Matrix Metalloproteinases (MMPs) Substrate Specificity + Structural Organization of secreted MMPs ECM Remodelling/Degrading Enzymes - MMPs – large class of proteases which can target ECM molecules - Some proteases have type 2 or type 3 repeats, but type 3 repeats found on lots of ECM molecules which explains why MMPs can attack more than one - Two types of MMPs: o Membrane type MMPs (MT-MMPs) which are activated intracellularly by furins in trans golgi network o Secreted Pro-MMPs which are activated by plasmin enzyme  Plasminogen = zymogen form of plasmin which can be activated by uPA (urokinase- plasminogen activator) or tPA (tissue plasminogen activator)  Serine Proteases  Plasmin, uPA, tPA (plasmin synonymous /w wound repair) - ADAM – A disintegrin and metaproteinase; two types exist: o Secreted ADAM-TS = ADAM /w at least one thrombospondin (TS) repeat (integrin motif found @ end of secreted form) o Membrane bound ADAMs form - Aminopeptidases (ANPs) – exopeptidases which chew proteins from ends - Cathepsins – cysteine proteases in lysosomes (terminal degradation, bring in partially degraded ECM molecules into lysosomes which cathepsins shred up) o Primarily lysosomal but small subset are transferred to PM where they can be active MMP substrates identified in complex biological samples by proteomic analysis: “degradomics” - Two main functions of MMPs o 1) remodel and degradation of ECM o 2)Silencing of Growth factor signalling and cytokines  Esp during embryogenesis, sheds ectodomains of receptors and integrins Pro-MMP-2 and MMP-9 - Cysteine switch is part of pro-domain, pre-peptide domain is lost in ER, secretion is as a pro-domain which reqs cleavage to be active - Catalytic domain has imp Zn2+ binding site and /w Hemopexin domain provide substrate specificity - Hemopexin domain is obligatory for collagenase activity of MT1 and imp for its dimerization - Removal of disintegrin of ADAMs = all adams would be called membrane type Activation of MMPs - inactive conformation has Cys-Zn2+ interactions removal of prodomain frees catalytic Zn2+ turning on MMP - Collagenases (e.g. MMP-1 and MT1-MMP) cleave interstitial T1/2/3C into characteristic 3/4 and 1/4 fragments /w a single cleavage site in c-terminus  imp in cancer cell metastasis  Cleaved collagens = gelatin Inhibition of MMPs by Tissue Inhibitors of Metalloproteinases (TIMPs) - TIMPs are the major endogenous regulators of MMP and ADAMs activities in Tissues - Four TIMPs identified: TIMPs1-4 - TIMPs bind to catalytic domain of MMPs @ 1:1 ratio - TIMP-1  soluble MMPs, but a poor inhibitor of MT-MMPs - TIMP-2,-3,-4  effective inhibitors of MT-MMPs - Dysregulation of TIMPs and MMPs = common scenario in cancer Dermal Wound Healing - Keratinocytes migrate into denuded area to re-epithelialize the wound. These cells migrate on a collagen substrate i.e. basal lamina. - Provisional matrix consisting of fibrin and plasma proteins is removed - Subcutaneous tissue fibroblasts (myofibroblasts) contract the ECM to facilitate wound closure - MMPs breakdown matrix in clots to create pathway for migrating keratinocytes o MMPs also involved in remodelling of matrix but unlike in embryogenesis, scar tissue is made - Inflammatory response aid in wound closure by convert normal fibroblast cells into contractile myofibroblasts cells that secrete more ECM Membrane Type MMPs (MT-MMPs) “Pericellular Proteolysis” - Role of MT1-MMP in cancer cell invasion - MT-MMPs are expressed during embryogenesis by mesenchymal cells, including fibroblasts, muscular cells, and osteoblasts - Expression decreases after birth but are reactivated when cells require remodelling of ECM e.g. wound healing, angiogenesis and cancer - Different than soluble MMPs bc they have TM domain (can be inhibited by TIMP2-4) o MT-MMPs also can be linked by GPI (glycosyl-phosphatidyl inositol) -anchoring signal - Main focus = MMP14/MT1-MMP (important in cancer process and basement membrane) Possible Mechanism for Localization and Turnover of MT1-MMP on cell surface - Spectrum of ECM proteins that can be cleaved by MT1-MMP o Native T1C,T2C,T3C (due to collagenase activity), FN, Laminin, Perlecan, Aggrecan o Non-ecm = CD-44 and Pro-TNFa (TNFa can promote peritoneal tumour cell metastasis Lec16) - MMP14/MT1-MMP gets together /w CD44 and promotes movement of this complex to leading edge of migrating cell (MT1-MMP/MMP14 comes onto cell surface already activated) - MT1-MMP is actually a dimer /w TIMP-2 attached to it to allow activity of just one MT1 o Although contradictory, since TIMP
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