A novel Intracellular activity for the serpin plasminogen activator inhibitor type-2

Darnell, Grant Andrew (2004). A novel Intracellular activity for the serpin plasminogen activator inhibitor type-2 PhD Thesis, School of Population Health, The University of Queensland.

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Author Darnell, Grant Andrew
Thesis Title A novel Intracellular activity for the serpin plasminogen activator inhibitor type-2
School, Centre or Institute School of Population Health
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Ass. Prof. Andreas Suhrbier
Ass. Prof. Toni Antalis
Total pages 187
Language eng
Subjects 1117 Public Health and Health Services
Formatted abstract  

Plasminogen activator inhibitor type 2 (PAI-2) is well documented as an inhibitor of the extracellular serine protease, urokinase-type plasminogen activator (uPA). This thesis identifies PAI-2 as having a novel intracellular function as a retinoblastoma protein (Rb) binding protein.

Analysis of intracellular PAI-2 expression found PAI-2 localised within both the cytoplasm and the nucleus. PAI-2 in the nucleus was found to co-localise with Rb by confocal microscopy, and Rb bound to PAI-2 could be immunoprecipitated from the nucleus with an anti-PAI-2 antibody. Although PAI-2 contained a consensus LXCXE Rb binding motif, the C-D interhelical region of PAI-2 was primarily responsible for binding Rb within the C-pocket. The C-D interhelical region of PAI-2 contained a novel Rb-binding motif, termed the PENF homology motif, which was also identified in many other cellular and viral Rb-binding proteins that also bound Rb in an LXCXE independent manner within the C-pocket (Chapter 2).

PAI-2 expression was shown to increase Rb protein levels post-transcriptionally by inhibiting Rb protein turnover. The PAI-2-mediated protection of Rb, and the subsequent increase in protein levels was shown to enhance Rb-mediated activities such as inhibition of E2F-1-mediated gene transcription and cell cycle arrest. The ability of PAI-2 to inhibit Rb turnover was shown to be dependent on both the C-D interhelical region and the reactive site loop (RSL) of PAI-2, demonstrating that both Rb binding and protease inhibition respectively are required for the new intracellular function of PAI-2. Incubation of cells with a specific inhibitor of the cysteine protease calpain-1 increased Rb levels, implicating calpain-1 in Rb turnover. Furthermore, calpain-1 was shown to cleave a small fragment from the C-terminus of Rb, and this cleavage could be inhibited by PAI-2 (Chapter 3).

PAI-2 expression in human papillomavirus (HPV) transformed cell lines protected Rb from the accelerated degradation mediated by the HPV E7, leading to recovery of Rb levels. Rb was then shown to repress transcription of the HPV oncoproteins E6 and E7 by inhibiting the transcription from the HPV-16 and HPV-18 URR promoter regions, allowing for the recovery of other E6 and E7 targeted proteins such as p53, c-Jun, and c-Myc. Rb-mediated repression of the HPV-18 URR was shown to be due to disruption of the binding of the C/EBPβ- YY1 dimer to the switch region within the HPV-18 URR. This allowed the dominant negative C/EBPP isoform, LIP to bind to the switch region and repress E6/E7 transcription. PAI-2 was also able to repress the HPV-16 URR; however, this activity was only partially dependant on Rb expression. Regulation of the HPV-URR by PAI-2 and Rb represents a novel cellular mechanism for the regulation of E6/E7 expression, and suggests a potential therapeutic role for PAI-2 against HPV transformed tumours and lesions (Chapter 4).

PAI-2 expression was shown to influence replication of the human immunodeficiency virus type-1 (HIV-1). PAI-2 expression increased HIV-1 replication 3-4 fold in Jurkat T-cells, THP-1 monocytes and HeLa epithelial cells. PAI-2 increased HIV replication by enhancing activity from the HIV-1 LTR core enhancer region in a NF-KB independent manner. Up-regulation of HIV-1 LTR driven transcription was again shown to be dependant on both the C-D interhelical region and protease inhibitory RSL of PAI-2. The PAI-2 mediated increase in Rb levels appeared to sequester E2F-1 away from the LTR core enhancer region, thereby alleviating E2F-1-mediated down-regulation of HIV-1 LTR driven transcription. The PAI-2-mediated increase of HIV-1 replication suggests that PAI-2 expression may be associated with enhanced HIV-1 replication in macrophages in vivo (Chapter 5).

The novel intracellular function described in this thesis for PAI-2 as an Rb binding protein begins to explain many of the diverse, uPA-independent phenotypes conferred by PAI-2 expression.

Keyword Serpins
Macrophages -- Activation
Genetic regulation

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Created: Tue, 08 Nov 2011, 14:03:52 EST by Ning Jing on behalf of The University of Queensland Library