Year

2011

Degree Name

Doctor of Philosophy

Department

University of Wollongong. School of Biological Sciences

Abstract

Overexpression of the plasminogen activation system has been found to play an

important role in the progression of a number of forms of cancer. Plasminogen is

activated to plasmin via the actions of the serine proteases urokinase plasminogen

activator (uPA) and tissue plasminogen activator (tPA).Whilst cell surface bound

plasminogen is protected from direct inhibition, uPA and tPA are inhibited by

plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2). PAI-1 and PAI-2

belong to the serpin protein superfamily (SerpinE1 and SerpinB2, respectively)

which, despite significant variations in primary sequence fold into a well conserved

structure consisting of three _–sheets and nine _-helices. PAI-2 is unique among the

serpins in containing a 33-amino acid interhelical loop between _-helices C and D

(the CD-loop), which is commonly removed in recombinant PAI-2 production. Upon

inhibition of cell bound uPA (or tPA), plasminogen activator:inhibitor complexes are

endocytosed by members of the low-density lipoprotein receptor (LDLR) family.

Tumour overexpression of uPA and PAI-1 correlates with poor prognosis and

increased metastatic potential. Conversely, tumour expression of PAI-2 (in

combination with uPA) is associated with favourable outcome and relapse free

survival. Previous work has suggested that the distinct binding interactions of PAI-1

and PAI-2, due to the presence of a high-affinity LDLR binding motif in PAI-1 that is

absent in PAI-2, may underlie differential downstream cellular behaviours.

The aims of this thesis were to: (1) Determine the impact that removal of the CD-loop

of PAI-2 had on uPA inhibition and clearance and confirm that this PAI-2 form (PAI-

2 _CD-loop) could be utilised in the further aims of this study; (2) characterise the

molecular basis underlying the differential interactions of PAI-1 and PAI-2, both

individually and in complex with uPA, with receptors of the LDLR family; and (3)

XV

examine the functional consequences resulting from the complementation of LDLR

binding in PAI-2, specifically the initiation of promitogenic signalling and cell

proliferation.

Removal of the CD-loop of PAI-2 was shown to have no impact on the inhibition of

both solution phase and cell surface uPA or on the rate of clearance of uPA from the

surface of MCF-7 cells. Additionally, uPA:PAI-2 _CD-loop had similar binding

kinetics (KD ~ 5 nM) for the very low-density lipoprotein receptor (VLDLR) to that

previous published for uPA:PAI-2 complexes. Furthermore, PAI-2 _CD-loop

expressed and purified in the system described here gave both higher yield and purity

than wild-type PAI-2 expressed and purified under identical conditions.

Introduction of the LDLR minimal binding motif present in the _-helix D of PAI-1 in

PAI-2 via site-directed mutagenesis enabled high affinity binding to VLDLR. The

binding of this mutant, PAI-2YK in complex with uPA was remarkably similar to that

of uPA:PAI-1 in both mechanism and affinity (heterogeneous analyte, KD values of

1.35 nM & 70.1 nM). Interestingly, mutation of an additional residue adjacent to the

LDLR binding motif was required for high affinity binding of PAI-2 to low-density

lipoprotein receptor related protein-1 (LRP1).

Complementation of the LDLR binding motif in PAI-2 had profound impacts on the

downstream functionality of PAI-2. Specifically, the LDLR-binding PAI-2 forms

enhanced uPA endocytosis on both VLDLR and LRP1 expressing cell lines, demonstrating that LDLR affinity is the critical determinant in the rate of uPA internalisation. Furthermore, treatment of MCF-7 cells with PAI-2 YK sustained ERK activation, and increased proliferation in a manner indistinguishable from the effects of PAI-1. XVI

The findings presented in this thesis clearly demonstrate that introduction of an otherwise absent LDLR minimal binding motif in PAI-2 increases affinity of uPA:PAI-2 complex for LDLRs to mimic that of uPA:PAI-1. This has a profound impact on the downstream function of PAI-2 via sustained activation of cell signalling pathways and subsequent proliferative effects. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these proteins in cancer.

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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.