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A Systematic Analysis of Nuclear Heat Shock Protein 90 (Hsp90) Reveals A Max Planck Institute of Immunobiology und Epigenetics Freiburg im Breisgau A systematic analysis of nuclear Heat Shock Protein 90 (Hsp90) reveals a novel transcriptional regulatory role mediated by its interaction with Host Cell Factor-1 (HCF-1) Inaugural-Dissertation to obtain the Doctoral Degree Faculty of Biology, Albert-Ludwigs-Universität Freiburg im Breisgau presented by Aneliya Antonova born in Bulgaria Freiburg im Breisgau, Germany March 2019 Dekanin: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Referent: Dr. Ritwick Sawarkar Koreferent: Prof. Dr. Rudolf Grosschedl Drittprüfer: Prof. Dr. Andreas Hecht Datum der mündlichen Prüfung: 27.05.2019 ii AFFIDAVIT I herewith declare that I have prepared the present work without any unallowed help from third parties and without the use of any aids beyond those given. All data and concepts taken either directly or indirectly from other sources are so indicated along with a notation of the source. In particular I have not made use of any paid assistance from exchange or consulting services (doctoral degree advisors or other persons). No one has received remuneration from me either directly or indirectly for work which is related to the content of the present dissertation. The work has not been submitted in this country or abroad to any other examination board in this or similar form. The provisions of the doctoral degree examination procedure of the faculty of Biology of the University of Freiburg are known to me. In particular I am aware that before the awarding of the final doctoral degree I am not entitled to use the title of Dr. Aneliya Antonova iii ACKNOWLEDGEMENTS First of all, I would like to express my gratitude to my supervisor Dr Ritwick Sawarkar for giving me the opportunity to carry out this challenging project in his lab. Thank you for guiding my way from a graduate to an independent and responsible scientist. I learned a lot from you! I am very grateful to all the former and present members of the lab for their scientific and moral support. Dear Parul, thank you for being such a supportive partner in that tough journey, a great friend and a valuable example. Barbara, Prashant, Fernando, Sergio, Erik, and Rebecca, I am glad I met every single one of you. I wish to thank the members of my thesis committee Prof Dr Rudolf Grosschedl and Prof Dr Andreas Hecht for their guidance. I would also like to acknowledge Dr Magdalena Baer- Rademacher, Dr Monika Lachner, Dr. Kyle Austin and Lisa Breitner, the coordinators of the International Max Planck Research School for Molecular and Cellular Biology (IMPRS- MCB) for always being there to help. I would like to thank my family and friends, who were so supportive and motivating during these years. My very special thanks go to my husband Nikolay Antonov who was always there to share my excitement in good times and hold my hand in bad days. Thank you for believing in me! And finally, thank you my little Thoma for being the sunshine in my days. I cannot wait to hear you saying “My mom is a scientist”. iv CONTENTS CONTENTS List of figures………………………………………………………………………………..viii List of tables…………………………………………………………………………………...x Abbreviations…………………………………………………………………………………xi Abstract……………………………………………………………………………………...xiii Zusammenfassung…………………………………………………………………………...xv CHAPTER I Systematic analysis of nuclear heat shock protein 90 (Hsp90) 1. Introduction ....................................................................................................................................... 1 1.1. Molecular chaperones ..................................................................................................... 1 1.2. Hsp90 .............................................................................................................................. 3 1.2.1. Structure and conformational cycle .......................................................................... 4 1.2.2. Function .................................................................................................................... 4 1.2.3. Regulation of Hsp90 ................................................................................................. 7 1.2.4. Chaperone cycle ..................................................................................................... 13 1.2.5. Hsp90 isoforms ....................................................................................................... 15 1.1.6. Hsp90 and cancer ................................................................................................... 16 1.2.7. Interactome studies ................................................................................................. 18 1.2.9. Nuclear Hsp90 ........................................................................................................ 23 1.2.9. Challenges and open questions ............................................................................... 27 2. Aims .............................................................................................................................................. 30 2.1. Nuclear Hsp90α and Hsp90β interactomes in human cells. .......................................... 30 2.2. Nuclear Hsp90α and Hsp90β PTMs. ............................................................................. 30 3. Materials and Methods .................................................................................................................. 31 3.1. Cell Culture and Cell Lines ........................................................................................... 31 3.2. Antibodies ..................................................................................................................... 31 3.3. Cell Fractionation .......................................................................................................... 31 3.4. SDS-PAGE .................................................................................................................... 33 3.5. Silver staining ................................................................................................................ 33 v CONTENTS 3.6. Western Blotting ........................................................................................................... 34 3.7. Nuclear Protein Affinity Purification coupled to Mass Spectrometry .......................... 34 3.7.1 Stable Cell Lines Generation ................................................................................... 34 3.7.2 Nuclear Protein Extract Preparation ........................................................................ 35 3.7.3 Protein Affinity Purification .................................................................................... 35 3.7.4. Protein Digestion and Mass Spectrometry Analysis .............................................. 36 3.8. SILAC labelling ............................................................................................................ 37 3.9. Di-Gly Immunoprecipitation ......................................................................................... 37 3.10. RNA isolation .............................................................................................................. 38 3.11. Immunofluorescence ................................................................................................... 38 3.12. Calcium Phosphate Transfection ................................................................................. 39 3.13. Synthetic Lethality Screen .......................................................................................... 39 4. Results ............................................................................................................................................. 40 4.1. Nuclear Hsp90 physical interactome investigated by affinity purification ................... 40 4.1.1. Nuclear Hsp90α interactions in normal growth conditions .................................... 42 4.1.2. Nuclear Hsp90β interactions in normal conditions ................................................ 51 4.1.3. Nuclear Hsp90α and Hsp90β interactions in stress conditions .............................. 53 4.2. Nuclear Hsp90 interactome investigated by revealing Hsp90-dependent ubiquitylated proteome ............................................................................................................................... 56 4.3. Hsp90 functional interactome investigation .................................................................. 72 4.4. Hsp90 PTMs .................................................................................................................. 73 4.5. Summary ....................................................................................................................... 75 5. Discussion and Outlook ................................................................................................................ 77 5.1. Nuclear Hsp90 interaction network ............................................................................... 77 5.1.1. Collaboration with nuclear protein folding and degradation machineries ............. 77 5.1.2. Role in gene expression regulation......................................................................... 80 5.1.3. RNA metabolism .................................................................................................... 81 5.1.3. Novel opportunities of anti-cancer therapies .......................................................... 84 5.1.4. Hsp90 during
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