ANNA OKSANEN Proprotein Convertase Enzymes FURIN and PCSK7 in Immune Regulation ACADEMIC DISSERTATION To be presented, with the permission of the Board of the BioMediTech of the University of Tampere, for public discussion in the Small Auditorium of Building M, Pirkanmaa Hospital District, Teiskontie 35, Tampere, on May 29th, 2015, at 12 o’clock. UNIVERSITY OF TAMPERE ANNA OKSANEN Proprotein Convertase Enzymes FURIN and PCSK7 in Immune Regulation Acta Universitatis Tamperensis 2055 Tampere University Press Tampere 2015 ACADEMIC DISSERTATION University of Tampere, BioMediTech Finland Supervised by Reviewed by Docent Marko Pesu Docent Petteri Arstila University of Tampere University of Helsinki Finland Finland Docent Zhi Jane Chen University of Turku Finland The originality of this thesis has been checked using the Turnitin OriginalityCheck service in accordance with the quality management system of the University of Tampere. Copyright ©2015 Tampere University Press and the author Cover design by Mikko Reinikka Distributor: [email protected] https://verkkokauppa.juvenes.fi Acta Universitatis Tamperensis 2055 Acta Electronica Universitatis Tamperensis 1547 ISBN 978-951-44-9802-2 (print) ISBN 978-951-44-9803-9 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://tampub.uta.fi Suomen Yliopistopaino Oy – Juvenes Print Tampere 2015 441 729 Painotuote “I need to write down my observations. Even the tiniest ones; they are the most important.” -Tove Jansson (1914-2001) CONTENTS LIST OF ORIGINAL COMMUNICATIONS ............................................................. 7 ABBREVIATIONS ............................................................................................................ 8 ABSTRACT........................................................................................................................ 11 TIIVISTELMÄ .................................................................................................................. 13 1 INTRODUCTION ....................................................................................................... 15 2 REVIEW OF THE LITERATURE........................................................................... 17 2.1 The immune system ............................................................................................... 17 2.2 CD4+ T helper cells ............................................................................................... 20 2.2.1 T helper cell differentiation ........................................................................... 20 2.2.2 T helper cell function ...................................................................................... 24 2.3 Biology of the proprotein convertase (PCSK) enzymes ................................... 26 2.3.1 The PCSK family ............................................................................................. 27 2.3.2 Tissue expression and subcellular localization of PCSKs ......................... 28 2.3.3 Substrates of PCSKs ....................................................................................... 30 2.3.4 Interaction studies on PCSKs ....................................................................... 32 2.4 PCSKs in health and disease ................................................................................. 33 2.4.1 Animal models and human patients ............................................................. 34 2.4.2 PCSKs in regulating the immune system ..................................................... 38 2.4.3 PCSKs in the pathogenesis of atherosclerosis ............................................ 42 2.4.4 Therapeutic potential of targeting PCSKs ................................................... 43 2.5 Studying the function of PCSKs in zebra fish ................................................... 44 3 AIMS OF THE STUDY .............................................................................................. 47 4 MATERIALS AND METHODS .............................................................................. 48 4.1 Experimental animals (I-II) .................................................................................. 48 4.2 Human subjects (III) ............................................................................................. 48 4.3 Antibodies (I-III).................................................................................................... 49 4.4 Plasmid constructs and cloning (I, II) ................................................................. 50 4.5 Cell culture, transfection, and generation of stable cell lines (I-II) ................ 50 4.6 Cell purification, activation, and flow cytometry (I, III) .................................. 51 4.7 Biochemical analysis of proteins (I-III) .............................................................. 52 4.7.1 Cell lysis and Western blotting (I-III) ........................................................... 52 4.7.2 Strep-tag affinity purification and mass spectrometry (I) .......................... 53 4.7.3 Co-immunoprecipitation and co-immunofluorescence (I) ....................... 54 4.8 F-actin polymerization assay (I) ........................................................................... 54 4.9 Cytokine measurements (I, III) ............................................................................ 55 4.10 Quantitative real-time PCR, Q-RT-PCR (I-III) .............................................. 55 4.11 Statistical analyses (I-III) ..................................................................................... 56 4.12 Ethical considerations (I-III) .............................................................................. 56 5 SUMMARY OF THE RESULTS .............................................................................. 58 5.1 FURIN is a central regulator of T cell-mediated immunity in mouse (I) ...... 58 5.1.1 FURIN is critical for host resistance against Toxoplasma gondii ............ 58 5.1.2 Th1/Th2 balance is affected by FURIN deficiency ................................... 59 5.1.3 FURIN inhibits IL-4Rα surface expression on naïve Th cells ................. 60 5.1.4 RAC/DOCK2 is a novel FURIN interacting complex in T cells ............ 61 5.2 PCSK7 is essential for zebra fish development and TGFβ1a regulation (II) 62 5.2.1 Embryogenesis and immune system gene expression are affected by PCSK7 silencing ........................................................................................................ 63 5.2.2 PCSK7 regulates the expression and proteolytic cleavage of TGFβ1a ... 64 5.3 FURIN is upregulated in human atherosclerotic plaques (III) ........................ 65 5.3.1 Expression of FURIN is induced upon immune cell activation .............. 66 5.3.2 BAFF and APRIL levels are not elevated in atherosclerotic patient serum .......................................................................................................................... 66 6 DISCUSSION ................................................................................................................ 68 6.1 PCSKs mediate both redundant and unique immunomodulatory functions ......................................................................................................................... 68 6.2 Expression of FURIN is critical for immune cell function .............................. 71 6.3 Interaction studies reveal new concepts of PCSK-mediated cellular regulation ........................................................................................................................ 72 7 CONCLUSIONS AND FUTURE PERSPECTIVES ............................................ 76 8 ACKNOWLEDGEMENTS ....................................................................................... 78 9 REFERENCES .............................................................................................................. 81 10 ORIGINAL COMMUNICATIONS ....................................................................... 99 LIST OF ORIGINAL COMMUNICATIONS This thesis is based on the following original publications, which are referenced in the text by their Roman numerals (I-III): I Oksanen A, Aittomäki S, Jankovic D, Ortutay Z, Pulkkinen K, Hämäläinen S, Rokka A, Corthals GL, Watford WT, Junttila I, O'Shea JJ, Pesu M. Proprotein Convertase FURIN Constrains Th2 Differentiation and Is Critical for Host Resistance against Toxoplasma gondii. J Immunol 2014;193(11)5470-9. II Turpeinen H*, Oksanen A*, Kivinen V, Kukkurainen S, Uusimäki A, Rämet M, Parikka M, Hytönen VP, Nykter M, Pesu M. Proprotein Convertase Subtilisin/Kexin Type 7 (PCSK7) Is Essential for the Zebrafish Development and Bioavailability of Transforming Growth Factor β1a (TGFβ1a). J Biol Chem 2013;288(51)36610-23. III Turpeinen H, Raitoharju E, Oksanen A, Oksala N, Levula M, Lyytikäinen LP, Järvinen O, Creemers JW, Kähönen M, Laaksonen R, Pelto-Huikko M, Lehtimäki T, Pesu M. Proprotein convertases in human atherosclerotic plaques: the overexpression of FURIN and its substrate cytokines BAFF and APRIL. Atherosclerosis 2011;219(2)799-806. *) Equal contribution The original publications have been reproduced with the permission of the copyright holders. 7 ABBREVIATIONS ADAM a disintegrin and metalloproteinase APC antigen presenting cell APRIL tumor necrosis factor ligand superfamily member 13 BACH2 BTB and CNC homology 2 BAFF tumor necrosis factor ligand superfamily member 13B BCR B cell receptor BMP bone morphogenetic protein CD cluster of differentiation CHD coronary heart disease CD4cre-furf/f