ECM PROTEASES IN INFLAMMATION, FIBROSIS & TUMORIGENESIS

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DESCRIPTION: Secreted metalloproteases play a leading role in processes of tissue remodeling by initiating the degradation of extracellular matrix (ECM). This proposal outlines the plan for investigating the biological function of these proteases. The experiments described in this application are based on the hypothesis that there is interaction with fixed molecular structures on the cell surfaces and/or fibrils of the ECM. Implicit in this hypothesis is the understanding that sequestering of enzymes at such sites is required for their physiological activation. Consequently, the net extracellular enzymatic activity depends on the availability of such sites and activation conditions. The mechanistic study of such interactions has been a focus of the investigator's attention, and just recently resulted is isolation of the novel membrane bound metalloprotease MT-MMP that is responsible for cell surface activation of 72 kDa type IV collagenase (72T4Cl). Elucidation of the activation mechanism of 72T4Cl substantiates an emerging concept that metastatic invasion requires epithelial-mesenchymal cooperation. Matrix metalloproteases are expressed in vivo by stromal cells. Epithelial tumor cells express protease receptor(s) on the cell surface. Interaction between these leads to activation that is highly specific and localized, thus presenting a new target for drug development. Studies of structure-function relationships in the human 72T4Cl, particularly the function of the carboxyl-end hemopexin-like domain, will be a focus for understanding of mechanisms of enzyme activation and interaction with the specific inhibitor TIMP2. Combination of site directed mutagenesis with determination of the tertiary structure of this domain will be main approach in these studies. The investigator's success in the purification and crystallization of the recombinant carboxyl end domain will be instrumental for the success of these studies. Several promising avenues of investigation has been developed aimed at elucidation of cell surface activation mechanism of interstitial collagenase (Cl1). A newly designed experimental immunofluorescence in vitro to detect the distribution of exogenously added purified enzymes in tissue culture will be instrumental in studies of interstitial collagenase interaction with cell surface and in the development of specific competitive ligands. Data obtained from these studies will provide an understanding of the molecular mechanisms involved in regulated extracellular proteolysis and the role of ECM metalloproteases in morphogenesis wound healing and numerous pathologic conditions.