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

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posted on 2024-11-11, 13:55 authored by Darren Neil Saunders
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.

History

Year

2000

Thesis type

  • Doctoral thesis

Faculty/School

Department of Biological Sciences

Language

English

Disclaimer

Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.

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