The overall goal of this project is to understand the special regulatory properties and the inhibitory specificity of plasminogen activator inhibitor type 1 (PAI-1). PAI-1 is an important regulator of the fibrinolytic cascade and has been implicated in the regulation of the tissue remodeling processes of neural growth, development, wound repair and cancer. PAI-1 is a member of the serine protease inhibitor (serpin) family, but unlike other serpins, it inactivates spontaneously with a half-life of 2 hours under physiological conditions. This occurs in the absence of any proteolytic cleavage events or chemical modifications, and the inactive or """"""""latent"""""""" conformation is a well-folded and highly ordered protein. The three-dimensional structure of this latent conformation of PAI-1 was determined by the principal investigator and Dr. Goldsmith. Mutagenesis experiments based on this model were performed to study the interaction of PAI-1 with plasminogen activators. It is proposed to continue using site- directed mutagenesis together with additional structural studies to ascertain the structural basis of the latent conformation and of the inhibitory specificity of PAI-1.
The specific aims are: (1) Perform mutagenesis of specific residues in PAI-1 to stabilize the active form of the protein and prevent or slow the transition into the latent conformation. The proposed mutagenesis is based on molecular modelling of the latent structure of PAI-1 that allows the investigator to predict regions of the protein and specific amino acid residues involved in the relative instability of PAI-1 in comparison to other serpins. The PAI-1 variants will be characterized by both biochemical and biophysical techniques to assess the degree of stabilization of the active form and learn how this inactivating transition is regulated. (2) Find sequences outside the reactive center that determine inhibitor specificity. Site- directed mutagenesis will be used to produce variant PAI-1 proteins that have altered specificity for serine proteases. (3) Produce large quantities of stable active PAI-1 and carry out crystallization trials on both the free inhibitor and PAI-1 in covalent complex with the catalytic domain of tPA.
| Tucker, H M; Gerard, R D (1996) Sequence requirements in the reactive-center loop of plasminogen-activator inhibitor-1 for recognition of plasminogen activators. Eur J Biochem 237:180-7 |
| Goldsmith, E J; Sheng-Cheng, C; Danley, D E et al. (1991) Preliminary X-ray analysis of crystals of plasminogen activator inhibitor-1. Proteins 9:225-7 |
