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Ribosomal Accuracy in Higher Eukaryotes – Mistranslation and Unfolded Protein Response

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Ribosomal Accuracy in Higher Eukaryotes – Mistranslation and Unfolded Protein Response Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Ribosomal accuracy in higher eukaryotes – mistranslation and unfolded protein response Meyer, Martin Dominique Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-120646 Dissertation Published Version Originally published at: Meyer, Martin Dominique. Ribosomal accuracy in higher eukaryotes – mistranslation and unfolded protein response. 2015, University of Zurich, Faculty of Science. Ribosomal Accuracy in Higher Eukaryotes – Mistranslation and Unfolded Protein Response Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Martin Dominique Meyer von Eschenbach / LU Promotionskomitee Prof. Dr. Erik C. Böttger (Vorsitz) Prof. Dr. Leo Eberl Prof. Dr. Vikram Panse Prof. Dr. Peter Sander Zürich, 10.09.2015 1 ACKNOWLEDGEMENTS .................................................................................... 1 2 SUMMARY ........................................................................................................... 3 3 ZUSAMMENFASSUNG ....................................................................................... 5 4 INTRODUCTION .................................................................................................. 7 4.1 The Ribosome ............................................................................................. 7 4.1.1 Architecture of the Ribosome .................................................................... 7 4.1.2 Mechanisms of Translation ....................................................................... 9 4.1.3 Ribosomal Accuracy ............................................................................... 12 4.1.4 Aminoglycosides ..................................................................................... 14 4.1.5 Aminoglycosides and Misreading ............................................................ 16 4.1.6 Protein Quality Control and Cellular Stress Response in Eukaryotes ..... 17 4.2 Mitochondria ............................................................................................. 22 4.2.1 Organization and Function ...................................................................... 22 4.2.2 Diseases Related to Mitochondrial Malfunction ...................................... 25 4.2.3 Mitochondrial Protein Import ................................................................... 27 4.2.4 Mitochondrial Quality Control and Unfolded Protein Response .............. 30 5 RESULTS........................................................................................................... 34 5.1 Aminoglycoside-Induced Misreading ..................................................... 34 5.1.1 Codon Selection for Gain-of-Function Assay .......................................... 34 5.1.2 In vitro System: Translation Inhibition ..................................................... 37 5.1.3 In vitro System: Misreading Induction ..................................................... 38 5.1.4 In vivo System: Translation Inhibition ...................................................... 42 5.1.5 In vivo System: Misreading Induction ...................................................... 45 5.1.6 Discussion ............................................................................................... 47 5.2 Drug-Induced Mitochondrial Unfolded Protein Response .................... 51 5.2.1 Test System and Drugs used .................................................................. 51 5.2.2 Mito-UPR: Validation of the system ........................................................ 55 5.2.3 Mito-UPR: Dose – Response .................................................................. 56 5.2.4 Mito-UPR: Induction of ClpP and Hsp60 ................................................. 57 5.2.5 Discussion ............................................................................................... 58 6 MATERIAL AND METHODS ............................................................................. 60 6.1 Cell Culture ............................................................................................... 60 6.2 Plasmids .................................................................................................... 60 6.2.1 Dual Luciferase Assay in-vitro ................................................................. 60 6.2.2 Dual Luciferase Assay in-vivo ................................................................. 61 6.3 Dual Luciferase Assay in-vitro ................................................................ 62 6.4 Dual Luciferase Assay in-vivo ................................................................. 62 6.5 β-Galactosidase Assay ............................................................................ 63 6.6 Mitochondrial in-organello Translation .................................................. 63 7 REFERENCES ................................................................................................... 65 8 XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death ................................................................................... 79 9 CURRICULUM VITAE ..................................................................................... 133 10 LIST OF PUBLICATIONS ................................................................................ 135 11 CONFERENCE PRESENTATIONS ................................................................. 137 12 SEMINARS AND COLLOQUIA ....................................................................... 138 1 ACKNOWLEDGEMENTS I would like to thank Prof. Dr. Erik C. Böttger for giving me the opportunity to work as a PhD student in his lab. You always had an open door- and ear policy for scientific questions and were very supportive in the cases of preparing scientific presentations and reports. Thanks to your critical reviews of my work I learned a great deal. I wish you many more successful years with interesting projects to come. I am very grateful to Dr. Dimitri Sherbakov and Dr. Rashid Akbergenov, postdocs in the Böttger group; they were always interested in my work and gave me useful advice on many occasions. I very much appreciate the fact that Prof. Dr. Leo Eberl, Prof. Dr. Vikram Panse and Prof. Dr. Peter Sander agreed to act as members of my thesis committee in the PhD program in Molecular Life Sciences and took the time to monitor my progress. A big thank you goes to the current and former members of the Böttger group; Stefan, Pietro, Heithem, Youjin, Pietro, Tanja J., and Thomas. All of you never hesitated to help me and offered me plenty of advice over the years. Also I would like to thank Susanna Salas for all of her help – for administrative objects and the many small but also important other things. In particular, special gratitude is reserved for Tanja Matt. Your friendship and love alone is already worth the years as PhD student! No matter what, I will always love you! My dear friends, I love you all and I am very happy to have you! Biggest shouts go especially to Dario, Felix and Sam! You had always an open ear for my stories straight from the lab bench, even when most of the time I was speaking about stuff you didn’t quite understand. Bringing me back down to earth or lifting me back up onto my feet when necessary, you always have been and always will 1 be there for me. I am tremendously thankful for having such reliable and loyal friends, you guys rock! Most importantly, I want to thank my family! Mom, Dad, you were always supportive on all issues and matters. Without you I wouldn’t be where I am today. I’d also like to thank my sister Danielle. I admire you for everything you do or did and I hope you stay the way you are! To my brother Roger; I deeply regret that I don’t know what you’re doing or where you are right now, but I won’t give up hope that we’ll see each other again soon. 2 2 SUMMARY The ribosome is a large macromolecular complex which mediates protein synthesis using messenger RNA (mRNA) as a template and aminoacyl-transfer RNA (tRNA) as a substrate. High accuracy of translation is essential for all living cells. Aminoglycoside antibiotics are used in clinical medicine to treat life- threatening infections with gram-negative bacteria. Aminoglycosides target the highly conserved core structure of the ribosome, in particular the A-site, where they inhibit protein synthesis and prevent proper mRNA decoding. The objective of this thesis was to analyze how aminoglycosides affect the human ribosome, i.e. to study aminoglycoside-induced translation inhibition and misreading. As a possible consequence of ribosomal misreading we investigated cellular unfolded protein responses, in particular in mitochondria (UPRmt) and the endoplasmatic reticulum (UPRER). The first part of the thesis examines how different aminoglycosides affect translation. Towards this end we established a gain-of-function assay to measure the efficacy of translational and ribosomal accuracy. This we did in-vitro using Rabbit Reticulocyte Lysate and in-vivo using HEK293 cells. We observed a significant correlation when comparing in-vitro vs. in-vivo data between translation inhibition or misreading induction. Geneticin was identified as the most potent misreading inducer and translation inhibitor of the cytoribosome. However, no direct correlation
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