The serine protease inhibitor superfamily (serpin) comprises a class of structurally related proteins that include many of the protease inhibitors found in blood, as well as proteins with unrelated or unknown functions. Serpins are involved in the regulation of such diverse processes as coagulation, fibrinolysis, complement activation, ovulation, angiogenesis, inflammation, neoplasia and viral pathogenicity. The serpins act as so-called """"""""suicide inhibitors,"""""""" reacting only once with their target proteinase to form a covalent complex. Serpins also interact with a number of nonproteinase ligands and these interactions can regulate serpin function either by directly modulating inhibitor activity or by restricting their action to a localized compartment. This proposal will focus on two closely related serpins, plasminogen activator inhibitor-I (PAI-1) and neuroserpin as models to investigate serpin function. We will build on extensive previous studies of PAI-1 and neuroserpin and use a combination of biochemical, structural and molecular approaches to address the structural basis of several important serpin interactions. We will identify, express and characterize specific mutations in vitro, and these will be used to map distinct structural regions of PAI-1 important for different functions, including inhibitory specificity. Specific hypotheses regarding the nature of the serpin inhibitory mechanism will also be tested. Methods will also be developed to obtain real time structural information and solution-phase protein dynamics of the serpin structure. These will include site-specific labeling and nuclear magnetic resonance (NMR) and site-specific excimer fluorescence. The interaction of PAl- 1 with members of the lipoprotein receptor family of clearance receptors will also be examined. Current methods of genetic selection will be refined to screen large numbers of PM-i clones for mutants with single functions specifically effected such as binding to the clearance receptors or with unique target proteinase specificity. Together, these studies should help to delineate many important PAI-1 functions and provide a better understanding of serpin function in general in both health and disease.
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