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Characterisation of Serpinb2 As a Stress Response Modulator University of Wollongong Research Online University of Wollongong Thesis Collection 1954-2016 University of Wollongong Thesis Collections 2015 Characterisation of SerpinB2 as a stress response modulator Jodi Anne Lee University of Wollongong Follow this and additional works at: https://ro.uow.edu.au/theses University of Wollongong Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorise you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of this work may be reproduced by any process, nor may any other exclusive right be exercised, without the permission of the author. Copyright owners are entitled to take legal action against persons who infringe their copyright. A reproduction of material that is protected by copyright may be a copyright infringement. A court may impose penalties and award damages in relation to offences and infringements relating to copyright material. Higher penalties may apply, and higher damages may be awarded, for offences and infringements involving the conversion of material into digital or electronic form. 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. Recommended Citation Lee, Jodi Anne, Characterisation of SerpinB2 as a stress response modulator, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2015. https://ro.uow.edu.au/theses/4538 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Characterisation of SerpinB2 as a stress response modulator A thesis submitted in fulfilment of the requirements for the award of the degree Doctor of Philosophy from University of Wollongong By Jodi Anne Lee Bachelor of Science (Honours 1) School of Biological Sciences University of Wollongong 2015 I Declaration This thesis is submitted in accordance with the regulations of the University of Wollongong in fulfilment of the higher degree in research of Doctorate of Philosophy. It does not contain any material previously published by another person except where due reference is made in the text. The experimental work described in this thesis is original and has not been submitted for a degree at any other University. Jodi Anne Lee - II - List of Publications Lee JA, Yerbury JJ, Farrawell N, Shearer RF, Constantinescu P, Hatters DM, Schroder WA, Suhrbier A, Wilson MR, Saunders DN, Ranson, M. SerpinB2 (PAI- 2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation. PLoS One 2015; 10(6). Epub 2015 June 18. PMC4470917. Yerbury JJ, Gower D, Vanags L, Roberts K, Lee JA, Ecroyd H. The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro. Cell Stress Chaperones 2013 Mar; 18(2):251-7. Epub 2012 Sep 21. PMID: 22993064 PMCID: PMC3581626 Vine KL, Chandran VI, Locke JM, Matesic L, Lee J, Skropeta D, Bremner JB, Ranson M. Targeting urokinase and the transferrin receptor with novel, anti-mitotic N-alkylisatin cytotoxin conjugates causes selective cancer cell death and reduces tumor growth. Curr Cancer Drug Targets. 2012 Jan 1;12(1):64-73. PubMed PMID: 22111834. Lee JA, Cochran BJ, Lobov S, Ranson M. Forty years later and the role of plasminogen activator inhibitor type 2/SERPINB2 is still an enigma. Semin Thromb Hemost. 2011 Jun;37(4):395-407. Epub 2011 Jul 30. Review. PubMed PMID: 21805446. De Souza M, Matthews H, Lee JA, Ranson M, Kelso MJ. Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand. Bioorg Med Chem. 2011 Apr 15;19(8):2549-56. Epub 2011 Mar 12. PubMed PMID: 21454081. Lee JA, Croucher DR, Ranson M. Differential endocytosis of tissue plasminogen activator by serpins SerpinE1 and SerpinB2 on human peripheral blood monocytes. Thromb Haemost. 2010 Dec;104(6):1133-42. Epub 2010 Sep 13. PubMed PMID: 20838737. Cochran BJ, Gunawardhana LP, Vine KL, Lee JA, Lobov S, Ranson M. The CD- loop of SerpinB2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity. BMC Biotechnol. 2009 May 14;9:43. PubMed PMID:19442270; PubMed Central PMCID: PMC2693429. - III - List of Presentations Poster Vine KL, Chandran VI, Locke JM, Matesic L, Lee J, Skropeta D, Bremner JB, and Ranson M. Targeting Urokinase and the Transferrin Receptor with Novel, Anti- Mitotic N-Alkylisatin Cytotoxin Conjugates Causes Selective Cancer Cell Death and Reduces Tumour Growth. 24th Lorne Cancer Conference, Lorne Victoria, Feb. 2012 Lee JA, Croucher DR and Ranson M. Differential endocytosis of Tissue Plasminogen Activator by Serpins PAI-1 and PAI-2 on human peripheral blood monocytes. Australian Society for Medical Research (NSW) Scientific Meeting. The Powerhouse Museum, Ultimo. (2010) Vine KL, Locke JM, Lee JA, Bremner, JB, and Ranson M. Synthesis and in vitro anticancer activity of a targeted plasminogen activator inhibitor type II (PAI-2) cytotoxin conjugate. 7th International Symposium On Targeted Anticancer Therapies, Amsterdam, The Netherlands, March 2009 Lee JA, Croucher DR and Ranson M The Plasminogen Activation System in Monocytic Cell Differentiation and Proliferation: potential target for plasminogen activation inhibitor type 2 based therapeutics. XIth International Workshop on Molecular & Cellular Biology of Plasminogen Activation, Saltsjöbaden, Sweden. (2007) Oral Lee JA, Saunders DN, Schroder W, Wilson M, Yerbury JJ, Marie Ranson. SerpinB2 (PAI-2) modulates protective cellular responses to proteotoxic stress and processing of intracellular protein aggregates. Plasminogen Activation System in Pathology Workshop, Illawarra Medical Research Institute, University of Wollongong NSW Australia. (2012) Lee JA, Croucher DR and Ranson M Cell surface interactions of Tissue Plasminogen Activator with peripheral blood monocytes. XIIth International Workshop on Molecular & Cellular Biology of Plasminogen Activation, Cold Spring Habor Laboratory, Cold Spring Harbor, New York. (2009) - IV - Acknowledgements I would like to sincerely thank Professor Marie Ranson, Dr Justin Yerbury and Dr David Croucher (Chapters 2 and 3) for the formal supervision of the work within this thesis. I will be forever grateful for the contributions they have made to the projects undertaken and for all that they have taught me. I would like to acknowledge and thank Dr Darren Saunders, Professor Mark Wilson, Dr Andrew Aquilina and Associate Professor Heath Ecroyd for the many lengthy discussions on proteastasis networks and experimental advice given, without which I would not have gotten so far. I would also like to thank Dr Wayne Schroder for supporting the working hypothesis for a role of SerpinB2 as a molecular chaperone and providing the mouse embryonic fibroblasts used within the latter part of this thesis, in which to test it. I will be forever grateful to Professor Robert Medcalf for believing me when I approached him in New York to discuss this work, in its infancy. I would like to thank all fellow PhD students whom I have worked alongside for the contribution they made to my health and wellbeing. I wish you all the best science can offer. - V - Abstract SerpinB2 [plasminogen activator inhibitor type-2 (PAI-2)] was first described as a placental protein, which regulated plasminogen activation via the inhibition of the urokinase (uPA) or tissue-type (tPA) plasminogen activators during pregnancy. Further work has confirmed that SerpinB2 is an efficient inhibitor of cell surface, receptor bound uPA and clearance of the serpin:protease complex occurs via members of the low density lipoprotein receptor (LDLR) family, specifically very low density lipoprotein receptor (VLDLR) and low density lipoprotein-related protein receptor (LRP). This property has allowed for the development of SerpinB2 drug conjugates for the selective targeting of uPA positive carcinoma cells. However, potential utility of SerpinB2 in haematological disorders, such as acute myeloid leukaemia (AML), as well as its role as a physiological inhibitor of tPA is not well characterised. To date biochemical analysis has shown that SerpinB2 is able to inhibit the two-chain (active) form of tPA. However, whether this inhibitory function occurs within the cellular context was unknown. This thesis investigates the potential use of exogenous SerpinB2 as a delivery vehicle for the specific targeting of lymphoma or leukaemia, via cell surface uPA using the relatively low versus high uPA expressing, monocytic cell lines U-937 and THP-1, respectively, as disease models. We show here that uPA:SerpinB2 is internalised via the Low Density Lipoprotein Receptor (LDLR) family members, specifically Low Density Lipoprotein-Related Protein (LRP) and Very Low Density - VI - Lipoprotein Receptor (VLDLR) in a uPA dependent manner and these findings are consistent with those previously reported for epithelial cancer cell lines. In order to clarify the role of SerpinB2 in the inhibition and clearance of tPA from the cell surface of monocytes and macrophages, we next assessed the formation and endocytosis of tPA:SerpinB2
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