The serine protease inhibitor superfamily 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. Despite their importance in the regulation of such diverse processes as coagulation, fibrinolysis, complement activation, ovulation, embryonic development, angiogenesis, inflammation, neoplasia and viral pathogenicity, a detailed understanding of the mechanism of serpin action is lacking. This application focuses on the structure-function correlates of plasminogen activator inhibitor-1 or PAI-1, which is considered one of the principal regulators of the plasminogen activator system. In addition to protease inhibition, PAI-1 interacts with a number of important ligands including vitronectin, heparin and lipoprotein receptor-related protein (LRP). Structurally, PAI-1 provides an excellent example of the general serpins ability to adopt multiple conformation states that include active, latent, oxidized and noninhibitory substrate forms. The proposal lists two broad specific aims.
In aim 1, experiments are proposed to investigate the structural and mechanistic basis of serpin function. In these studies, several new single Ser(Cys containing PAI-1 mutants will be constructed in (-strands 1A and 2A and helix F, which are potential areas of contact between PAI-1 and several ligands including vitronectin. Interaction of PAI-1 with enzymes in these regions will be monitored by reaction of the Cys residue to DTNB. Additional studies in this aim will attempt to identify a site or sites in PAI-1, distant from the reactive site, that are important for its interaction with TPA. These studies will utilize the noninhibitory PAI-1, P1-Ala mutant form (active and latent) that binds to TPA, and determine if this mutation competes with wild type PAI-1 for TPA binding and affects the second order rate of wild type PAI-1 inhibition of TPA. Positive results will lead to chemical crosslinking studies and the production of a random PAI-1, P-1-Ala mutant library to define the exosite location. Related studies in this aim will focus on the development of a solid phase binding assay for vitronectin and LRP using a 32P labeled PAI-1 preparation constructed with an N-terminal six residue peptide tag that is phosphorylated by heart muscle kinase. This 32P labeled preparation retains full biologic activity and binds vitronectin in a manner indistinguishable from wild type PAI-1. Finally, experiments are proposed in this aim to further characterize the interaction of PAI-1 with vitronectin using pure preparations of various PAI-1 conformers as well as PAI-1 in complex with UPA and thrombin. These studies are designed to test the hypothesis that loop-inserted forms of PAI-1 have reduced affinity for vitronectin.
In aim 2, experiments are proposed to test the hypothesis that PAI-1 contains a cryptic LRP binding site by assessing the affinity of 32P labeled PAI-1 for LRP, alone or in complex with different enzymes, using a solid phase binding assay. Subsequent studies will investigate the cellular interaction and degradation of PAI-1 and PAI-1-protease complexes by rat fetal type-II pneumocytes that surface express both LRP-1 and LRP-2. The LRP binding domain in PAI-1 would also be identified using an existing PAI-1 phage mutant library.
Showing the most recent 10 out of 26 publications
