Structure-function relationships in plasminogen activator inhibitor type-2 (PAI-2)

Plasminogen activator inhibitor type-2 (PAI-2) is a member of the serine protease inhibitor (serpin) protein family. The known primary biochemical targets of PAI-2 are tissue-type and urokinase-type plasminogen activators (tPA and uPA) and it has been implicated in a number of physiological/pathological processes include pregnancy, skin differentiation, inflammation, apoptosis and metastasis. The serpin inhibitory mechanism essentially involves kinetic trapping of the protease in a stable serpimprotease complex accompanied by significant conformational changes in the serpin molecule. Although a number of serpins have been extensively studied, the structural changes associated with inhibitory activity in PAI-2 were not well characterised. Biochemical characterisation (urea denaturation, fluorimetry, CD spectroscopy, inhibitory activity) and immunological analysis (using a monoclonal antibody (#2H5) specific for relaxed PAI-2) were used in this thesis to examine conformational changes associated with PAI-2 inhibitory activity. Results confirmed that the PAI-2 inhibitory mechanism involves cleavage of the Arg380-Thr381 (PI-PI') bond and insertion of the reactive centre loop (RCL) into β-sheet A, resulting in transition of the molecule to a more thermodynamically stable, relaxed (R) state. Furthermore, using a panel of synthetic RCL peptides it was shown that interactions in the proximal hinge region are crucial for RCL insertion in PAI-2. In particular, the formation of a hydrogen bond between the sidechains of Thr367 (P14) and the adjacent Tyr258 on strand 2 of β-sheet B appears to be a critical determinant of RCL insertion in PAI-2. This pairing is highly conserved in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity. MAb #2H5 provides a novel immunological probe for biologically relevant expression of PAI-2 inhibitory activity and has proven useful in localisation of in vivo sites where PAI-2 is actively inhibiting uPA or other target proteases, leading to a better understanding of the physiological roles of this molecule. Preliminary investigation of relaxed PAI-2 expression in U937 and HeLa cells did not provide definitive evidence for a specific mechanism of PAI-2 mediated protection from apoptosis. However, results indicate that it may be possible to use mAb #2H5 in further studies to identify potential intracellular targets of PAI-2.