The long-term goal of this project is to understand the molecular mechanisms that control extracellular matrix degradation (turnover) on the cell surface. A recently discovered human endothelial cell surface, membrane protease may be a key molecule involved in cell surface proteolysis.
The Specific Aims of this renewal application will focus on endothelial membrane proteases. 1) The structure and cell type specificity of the endothelial membrane proteases will be examined by isolating the molecule and producing monoclonal antibodies, particularly these directed against the catalytic site and the membrane proteases unique to endothelial cells. A full length cDNA to the endothelial membrane proteases will be isolated to determine whether the functional protease molecule is the product of a single gene and to examine the putative proenzymatic, catalytic, and transmembrane domains. Preliminary data indicate that activation of the endothelial membrane proteases appears to require cleavage of a propeptide and dimerization of their subunit polypeptides. 2) Expression of the cDNA in human cells that lack membrane proteases will be used to identify regions of amino acid sequences that determine the biological function of the protein. If active sites are indicated, site-specific mutagenesis of key amino acid residues will be carried out to elucidate the function of the endothelial membrane proteases in vitro in term of protease activation, dimerization, and catalytic site. 3) Integrin-fibronectin complexes are centrally involved in the mechanism of extracellular matrix assembly. Therefore, specific cleavage of the complexes by membrane proteases will be characterized and then various angiogenesis models will be utilized to examine in vivo whether modulation of membrane proteases plays a particularly important regulatory role in endothelial cell function by altering extracellular matrix assembly. 4) The role of endothelial membrane proteases in lung angiogenesis and embryogenesis will be examined in term of their expression and localization using angiogenesis models and during development of normal and fibrotic lungs. These biochemical and cellular approaches will help to elucidate both the positive and negative regulatory mechanisms that control normal tissue organization, repair, remodeling, and disease pulmonary fibrosis.
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