The long-term goal of this project is to understand the molecular mechanisms that control extracellular matrix degradation (turnover) on the cell surface of fibroblasts and endothelial cells during tissue repair and angiogenesis. A group of serine integral membrane proteases (seprase and dipeptidyl peptidase IV [DPPIV] are implicated in cell surface collagen proteolysis that occurs at the leading edge invadopodia of migratory fibroblasts and endothelial cells.
The specific aims of this renewal application will focus on determining the role(s) of seprase and DPIV in tissue repair and angiogenesis, as well as the identification of potential inhibitors for the protease localization and activation at invadopodia. Thus, (1) the role of seprase and DPPIV in cell activation for tissue remodeling will be determined by investigating the expression and formation of protease complexes on the surface of migrating cells on collagen fibers and/or in angiogenesis and wound-healing models. Variations in transfectant expression and inhibitory antibodies against seprase/DPPIV should reveal a very specific set of defects that will address the role of seprase and DPPIV in tissue remodeling. (2) The role of alpha3beta1 integrin in protease docking, localization, and activation at invadopodia will also be examined. This will be demonstrated by the biochemical characterization of protease-integrin association, as well as the determination of such association in living cells, using resonance energy transfer microscopy. (3) Inhibitor-substrate-, and alpha3beta- binding specificity of seprase will be determined using purified native or recombinant enzymes in conjunction with peptide-phage display technology and applications of available reagents, including chemically modified tetracyclines and potent inhibitors or substrate-peptides of proline-specific peptidases. Finally, (4) x-ray crystallography will determine structure or recombinant seprase at atomic resolution. The overall goal is to explore new approaches toward identifying potential inhibitor and substrate-binding molecules for cell surface seprase and its complexes as therapeutic agents in treatment wounds and controlling angiogenesis of human cancer, i.e., repairing the damage caused by cancer.
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