Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2007 Identification of novel protein interaction partners of the oxygen-sensing HIF prolyl-4-hydroxylase 1 (PHD1) and characterization of the interactor onconeural cerebellar degeneration-related protein 2 (Cdr2) Hofmann, Verena Simone Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163609 Dissertation Published Version Originally published at: Hofmann, Verena Simone. Identification of novel protein interaction partners of the oxygen-sensing HIF prolyl-4-hydroxylase 1 (PHD1) and characterization of the interactor onconeural cerebellar degeneration- related protein 2 (Cdr2). 2007, University of Zurich, Faculty of Science. Identification of Novel Protein Interaction Partners of the Oxygen-sensing HIF Prolyl-4-hydroxylase 1 (PHD1) and Characterization of the Interactor Onconeural Cerebellar Degeneration-related Protein 2 (Cdr2) Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Verena Simone Hofmann aus Deutschland Promotionskomitee Prof. Dr. Roland H. Wenger (Vorsitz) Dr. Gieri Camenisch (Leitung der Dissertation) PD Dr. Ingo Flamme Zürich, 2007 This work has been performed under the supervision of Dr. Gieri Camenisch and Prof. Dr. Roland H. Wenger at the Institute of Physiology and Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, CH-8057 Zürich, Switzerland Table of contents – Yeast two-hybrid screenings Abbreviations ........................................................................................................3 Zusammenfassung .............................................................................................5 Summary..............................................................................................................7 Introduction .........................................................................................................9 1 The hypoxia-inducible factor (HIF)..........................................................9 1.1 Discovery and function of HIF.................................................................9 1.2 Composition of the HIF complex...........................................................10 1.3 Regulation of HIF-1 by oxygen ...........................................................11 1.3.1 Oxygen-dependent regulation of HIF-1 protein stability......................11 1.3.2 Oxygen-dependent regulation of the HIF-1 transactivation activity.....12 1.4 Additional positive and negative regulators of HIF................................12 2 The HIF prolyl-4-hydroxylases function as oxygen sensors..................15 2.1 The HIF prolyl-4-hydroxylases (PHDs) .................................................15 2.2 PHD mRNA expression pattern............................................................16 2.3 Enzymatic activity and catalytic mechanism of the PHDs.....................16 2.4 Substrate specificity..............................................................................17 2.5 Regulation of PHD expression and degradation...................................18 2.6 Regulation of prolyl-4-hydroxylation activity..........................................19 2.6.1 Molecular oxygen..................................................................................19 2.6.2 Iron and divalent metal ions..................................................................20 2.6.3 Ascorbate..............................................................................................20 2.6.4 2-oxoglutarate.......................................................................................21 2.6.5 Nitric oxide (NO) ...................................................................................21 2.6.6 Reactive oxygen species (ROS)...........................................................21 2.7 Known interactors of the PHDs.............................................................22 3 The HIF prolyl-4-hydroxylase 1 – PHD1...............................................24 3.1 Cloning of PHD1...................................................................................24 3.2 Transcriptional regulation of PHD1.......................................................25 3.3 Alternative initiation of translation and proteolytic regulation of PHD1..26 3.4 Possible function of PHD1....................................................................27 4 Working hypothesis ..............................................................................28 Material and Methods .......................................................................................30 Results ..............................................................................................................44 1 Table of contents – The onconeural antigen Cdr2 Abstract ..............................................................................................................73 Introduction .......................................................................................................74 1 The onconeural antigen Cdr2...............................................................74 1.1 Cdr2 mRNA and protein expression pattern.........................................75 1.2 What is the physiological function of Cdr2 ?.........................................76 2 The role of Cdr2 in paraneoplastic neurological disorders (PNDs) .......80 2.1 Definition of PNDs ................................................................................80 2.2 Classification of PNDs..........................................................................80 2.3 Proposed pathogenesis of PNDs..........................................................81 2.4 Characteristics of PND-associated tumors ...........................................83 2.5 Immunology of PNDs............................................................................83 2.5.1 Antibodies in PND.................................................................................83 2.5.2 Cytotoxic T cells mediate the PND tumor immune response................84 2.5.3 Antigen cross-presentation by dendritic cells in PND tumor immunity ..85 2.5.4 CD4+ T cells determine CD8+ T cell activation or tolerance..................87 2.6 Paraneoplastic neurological degenerations: keys to tumor immunity ...87 Material and Methods .......................................................................................89 Results ............................................................................................................100 Discussion.......................................................................................................118 Conclusion and Outlook.................................................................................126 References........................................................................................................127 Acknowledgements...........................................................................................138 Curriculum vitae ................................................................................................139 2 Abbreviations 2-OG 2-oxoglutarate Ang2 angiopoietin-2 AKT Synonym for protein kinase B APC antigen-presenting cell ARD1 arrest-defective-1 ARNT aryl hydrocarbon receptor nuclear translocator ATF-4 activating transcription factor-4 bHLH basic helix-loop-helix BSA bovine serum albumin C-TAD carboxy-terminal transactivation domain Cdr cerebellar degeneration-related protein ChIP chromatin immunoprecipitation CITED2 cAMP-responsive element-binding protein (CBP)/p300- interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail CODDD c-terminal oxygen-dependent degradation domain CTL cytotoxic T lymphocyte DC dendritic cell DFX desferrioxamine EGL egg-laying abnormal EPO erythropoietin ERK extracellular signal-regulated protein kinase ER endoplasmatic reticulum FIH factor inhibiting HIF FKBP38 FK506-binding protein 38 GSE genetic supressor element GST glutathione-S-transferase HDM2 human ortholog of mouse Mdm2 HIF hypoxia-inducible factor HLZ helix-leucine zipper HPH HIF prolyl hydroxylase HRE hypoxia response element ICAM-1 intercellular adhesion molecule 1 IGF1/2 insulin-like growth factor 1/2 ING4 inhibitor of growth family member 4 Km Michaelis Menten constant NGF nerve growth factor NF-B nuclear factor kappa B NLS nuclear localization signal NODDD N-terminal oxygen-dependent degradation domain NO nitric oxide NOS nitric oxide synthase N-TAD amino-terminal transactivation domain MAPK mitogen-activated protein kinase MDM2 mouse double minute 2 MEF mouse embryonic fibroblast MHC major histocompatibility complex mTOR mammalian target of rapamycin MCS multiple cloning site OD optical density ODDD oxygen-dependent degradation domain p300 300 kDa protein, histone acetyl transferase PAS PER/ARNT/SIM PCD paraneoplastic cerebellar degeneration PER Drosophila periodic protein PDFG-B platelet-derived growth factor B PHD prolyl-4-hydroxylase domain protein PI3-K phosphatidylinositol 3-kinase 3 PKC protein kinase C PKB protein kinase B PND paraneoplastic neurological disease PTEN phosphatase and tensin homolog deleted on chromosome ten Siah seven in absentia homolog SIM Drosophila single-minded protein SLE systemic lupus erythematosus SUMO small ubiquitin-related modifier TAD transactivation domain TAP transporter associated with antigen processing