Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Multimodal regulation of the oxygen sensing pathway by presenilin membrane proteases Kaufmann, M R Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-69298 Dissertation Originally published at: Kaufmann, M R. Multimodal regulation of the oxygen sensing pathway by presenilin membrane proteases. 2012, University of Zurich, Faculty of Science. Multimodal Regulation of the Oxygen Sensing Pathway by Presenilin Membrane Proteases Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Muriel Rahel Kaufmann von Sursee LU Promotionskomitee Prof. Dr. Roland H. Wenger (Vorsitz) PD Dr. Gieri Camenisch (Leitung der Dissertation) Prof. Dr. Roger Nitsch Dr. Ester M. Hammond Zürich, 2012 This work has been performed under the supervision of PD 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 Table of Contents Summary ................................................................................................................................... 3 Zusammenfassung .................................................................................................................... 6 General Introduction: Hypoxia ...................................................................................................10 1 The Hypoxia-Inducible Factor (HIF) ................................................................................10 1.1 Structure and Expression ........................................................................................10 1.2 Regulation ...............................................................................................................12 2 The Prolyl-4-Hydroxylases..............................................................................................14 2.1 PHD1 ......................................................................................................................14 2.2 PHD2 ......................................................................................................................15 2.3 PHD3 ......................................................................................................................15 2.4 PHD-interacting Proteins .........................................................................................15 3 References .....................................................................................................................17 4 Aims of the Thesis ..........................................................................................................21 Project I: Presenilin Function in the Oxygen Sensing Pathway ..................................................21 1 Introduction ....................................................................................................................21 1.1 FKBP38 ...................................................................................................................21 1.2 Presenilins ...............................................................................................................24 2 Working Hypothesis........................................................................................................33 3 Original Publication ........................................................................................................34 4 Unpublished Data ...........................................................................................................63 4.1 Materials and Methods ............................................................................................63 4.2 Results ....................................................................................................................64 5 Discussion ......................................................................................................................72 6 References .....................................................................................................................79 Project II: Onconeuronal Cerebellar Degeneration-Related Antigen Cdr2 as a Novel Diagnostic Marker for Renal Cancer ...........................................................................................................88 1 Introduction ....................................................................................................................88 1.1 The Cerebellar Degeneration-Related Onconeural Antigen Cdr2 ............................88 2 Original Publication ........................................................................................................92 3 Manuscript in Review ................................................................................................... 120 4 Conclusion ................................................................................................................... 129 5 References ................................................................................................................... 131 1 TABLE OF CONTENTS Contributions to the Publications and Antibody Licensing ....................................................... 134 Appendix ................................................................................................................................. 136 1 Additional Manuscript ................................................................................................... 136 2 Abbreviations................................................................................................................ 157 Curriculum Vitae ..................................................................................................................... 159 Acknowledgements ................................................................................................................. 162 2 SUMMARY Summary This dissertation is divided into two parts. The first project addresses the role of presenilin 1 and 2 in the regulation of the oxygen sensing pathway. In the second part, we investigated a potential role for the onconeural cerebellar degeneration related antigen Cdr2 as a novel tumor marker in papillary renal cell carcinoma. The two projects will be described and discussed separately, with a general introduction about hypoxia at the beginning. The availability of free oxygen on Earth was one of the prerequisites for the evolution of eukaryotic life. Multicellular organisms need oxygen to efficiently produce ATP. Therefore, mechanisms for adequate adaptation to low oxygen environments (hypoxia) was developing during evolution. The main research interest of our group is to understand the molecular and cellular mechanism of sensing and adaptation to hypoxic environments. Hypoxia is a driving force in many physiological and pathophysiological processes such as high mountain climbing, red blood cell synthesis, wound healing, cancer progression and neurodegenerative disorders. The molecular sensors of hypoxia are the prolyl-4-hydroxylase domain containing proteins (PHDs) that regulate the degradation of the hypoxia-inducible factors (HIFs) via oxygen dependent hydroxylation. In normoxia, the von Hippel-Lindau tumor suppressor protein (pVHL) binds to hydroxylated HIF which leads to polyubiquitination and subsequent proteasomal destruction. In hypoxia, the cofactor oxygen is limited and the hydroxylation reaction is therefore reduced. The stabilized HIF subunit then translocates to the nucleus and forms a potent transcription factor together with its constitutive counterpart HIF. HIF regulates the expression of more than 200 genes that are involved in the adaptation to low oxygen partial pressure. Apart from oxygen- dependent HIF protein regulation, there is growing evidence that HIF mRNA is regulated by oxygen-independent mechanisms, for example by pro-inflammatory stimuli such as nitrogenmonoxide or cytokines. 3 SUMMARY Project I: Recently, we have shown that the peptidyl/prolyl cis/trans isomerase FKBP38 specifically interacts with PHD2 and negatively regulates its protein stability (Barth et al. 2007). A report suggesting that presenilin 1 and 2 (PSEN1/2) interact with FKBP38 and are involved in the negative regulation of FKBP38 protein levels (Wang et al. 2005) draw our attention to a potential role of PSEN1/2 in PHD/HIF signaling. Presenilin (PSEN) 1 and 2 are the catalytic components of the -secretase complex which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid beta (A) that is crucially involved in the pathogenesis of Alzheimer’s disease. We found that PSEN1/2-deficient mouse embryonic fibroblasts (MEFs) express more FKBP38 protein that leads to reduced constitutive PHD2 protein levels. While decreased PHD2 levels would predict an increased hypoxic response, we interestingly found the opposite. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts (MEFs) and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice and resulted in a blunted hypoxic induction of specific HIF-target genes. While HIF-1 protein stability was not altered, we found decreased HIF-1