Aneuploidy Impairs Protein Folding and Genome Integrity in Human Cells
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Aneuploidy impairs protein folding and genome integrity in human cells Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Neysan Donnelly, MSc Biochemie aus Galway, Irland Mai 2016 Eidesstaattliche Erklärung Hiermit erkläre ich an Eides statt, dass ich die vorliegende Dissertation selbstständig und ohne unerlaubte Hilfe angefertigt habe. Ich habe weder anderweitig versucht eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile derselben einer anderen Prüfungskomission vorgelegt. München, den ............................. ........................................................ (Unterschrift) Promotionsgesuch eingereicht am: Datum der mündlichen Prufung: Erster Gutachter: Prof. Dr. Stefan Jentsch Zweiter Gutachter: Prof. Dr. John Parsch 2 Table of Contents 1. Abbreviations.................................................................................................................... 5 2. List of publications ............................................................................................................ 8 3. Declaration of contribution as co-author............................................................................ 9 4. Summary .........................................................................................................................10 5. Zusammenfassung............................................................................................................11 6. Aims of the thesis.............................................................................................................13 7. Introduction .....................................................................................................................14 7.1 Causes of aneuploidy ..........................................................................................15 7.2 Models to study aneuploidy ................................................................................18 7.3 Consequences of aneuploidy ...............................................................................24 7.3.1 Immediate effects of chromosome mis-segregation ................................24 7.3.2 Chronic consequences of aneuploidy .......................................................26 7.4 Role of aneuploidy in human disease...................................................................31 7.4.1 Trisomy syndromes .................................................................................31 7.4.2 Role of aneuploidy in other conditions ....................................................32 7.4.3 Aneuploidy and aging..............................................................................33 7.4.4 Aneuploidy and cancer – friend or foe?....................................................33 7.5 The effects of aneuploidy on the proteostasis network ........................................35 7.5.1 The proteostasis network ........................................................................36 7.5.2 The role of the PN in aging and disease....................................................38 7.5.2 Effects of aneuploidy on proteostasis ......................................................41 7.6 The effects of aneuploidy on the maintenance of genome stability ......................42 7.6.1 Aneuploidy and numerical CIN – a two-way street? .................................42 7.6.2 Structural and whole-chromosomal aneuploidy .......................................43 7.6.3 Aneuploidy and single-nucleotide aberrations .........................................44 8. Results............................................................................................................................46 8.1 HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells ........................................................................................46 8.2 The presence of extra chromosomes leads to genomic instability .........................67 9. Discussion.......................................................................................................................94 9.1 Aneuploidy undermines cellular proteostasis by impairing protein folding ...........94 9.1.1 Consequences of the protein folding defect in aneuploid cells .................98 9.1.2 Implications of impaired proteostasis for cancer and trisomy syndromes ............................................................................................................................. 100 3 9.2 Effects of aneuploidy on genome stability.......................................................... 102 9.3 A link between impaired proteostasis and genomic instability in aneuploid cells? ......................................................................................................................................... 104 9.4 Implications of aneuploidy-induced genomic instability for disease .................... 105 9.5 Conclusions and perspectives ............................................................................ 106 10. References.................................................................................................................... 108 11. Acknowledgements....................................................................................................... 125 12. Curriculum vitae ........................................................................................................... 126 4 1. Abbreviations 4-NQO 4-nitroquinoline-N-oxide 17-AAG 17-N-allylamino-17-demethoxygeldanamycin 53BP1 p53 binding protein 1 AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide AIF Apoptosis-inducing factor AMPK 5' adenosine monophosphate-activated protein kinase APP Amyloid beta precursor protein ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate CDC6 Cell Division Cycle 6 CDC37 Cell Division Cycle 37 CDK2 Cyclin-dependent kinase 2 CDK4 Cyclin-dependent kinase 4 CDK6 Cyclin-dependent kinase 6 CENP-E Centromere-associated protein E CIN Chromosomal instability DDR DNA damage response DNA Deoxyribonucleic acid DS Down's syndrome DSB Double strand break DSCR Down syndrome critical region DSCR1 Down syndrome critical region gene 1 EGF Epidermal growth factor 5 EOAD Early onset Alzheimer's disease ESC Embryonic stem cell ESR Environmental stress response FANCA Fanconi anemia, complementation group A FANCD1/BRCA2 breast cancer 2/Fanconi anemia, complementation group D FISH Fluorescence in situ hybridization GDBH Gene dosage balance hypothesis HSF1 Heat Shock Factor 1 Hsp104 Heat Shock Protein 104 HSP27 Heat Shock Protein 27 HSP70 Heat Shock Protein 70 HSP90 Heat Shock Protein 90 HSR Heat Shock Response iPSC Induced pluripotent stem cell LC3 Microtubule-associated proteins 1A/1B light chain 3A MCAK Mitotic centromere-associated kinesin MCM Minichromosome maintenance protein complex MEF Mouse embryonic fibroblast MM Multiple myeloma MVA Mosaic variegated aneuploidy MYC V-Myc Avian Myelocytomatosis Viral Oncogene Homolog ORC1 Origin Recognition Complex, Subunit 1 ORC2 Origin Recognition Complex, Subunit 2 Pol ζ DNA polymerase ζ 6 PN Proteostasis network RNA Ribonucleic acid RNA Pol II RNA polymerase II ROS Reactive oxygen species RPA1 Replication Protein A1, 70kDa RPE-1 Retinal pigment epithelium cells SAC Spindle assembly checkpoint SNP Single nucleotide polymorphism TKNEO thymidine kinase with neomycin phosphotransferase reporter gene Ubp6 Ubiquitin carboxyl-terminal hydrolase 6 UPR Unfolded protein response v-Src Proto-oncogene tyrosine-protein kinase Src XIST X-inactive specific transcript XRCC1 X-ray repair cross-complementing protein 1 YAC Yeast artificial chromosome 7 2. List of publications Donnelly N, Passerini V, Dürrbaum M, Stingele S, Storchová Z. HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells. EMBO J. 2014 Oct 16;33(20):2374-87 Passerini V*, Ozeri-Galai E*, de Pagter M, Donnelly N, Schmalbrock S, Kloosterman WP, Kerem B, Storchová Z. The presence of extra chromosomes leads to genomic instability. Nat Commun. 2016 Feb 15;7:10754 *these authors contributed equally to this work 8 3. Declaration of contributions as co-author Donnelly N, Passerini V, Dürrbaum M, Stingele S, Storchová Z. HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells. EMBO J. 2014 Oct 16;33(20):2374-87 Neysan Donnelly contributed to this work by designing, planning and performing the experiments depicted in Figure 1, Figure 2 C,D, Figure 3 and Figure 4 and corresponding supplementary figures. In addition, he participated in the creation of figures, the interpretation and discussion of results, as well as in the writing of the paper. Passerini V*, Ozeri-Galai E*, de Pagter M, Donnelly N, Schmalbrock S, Kloosterman WP, Kerem B, Storchová Z. The presence of extra chromosomes leads to genomic instability. Nat Commun. 2016 Feb 15;7:10754 *these authors contributed equally to this work Neysan Donnelly contributed to this work by planning and performing the experiments shown in Figure 5 E and Figure 6 D-F. He also participated in the interpretation and discussion of results. Martinsried, den...................... Neysan Donnelly Zuzana Storchová ..................................... ....................................... 9 4. Summary Aneuploidy or imbalanced chromosome content is the cause of pathological conditions such as Down's syndrome and is also a hallmark of cancer where it is linked with malignancy