Aneuploidy causes proteotoxic stress in Saccharomyces cerevisiae. ARtCHNEU By MASSACHUSCE Ana Belen Oromendia B.S. Biochemistry University of Minnesota- Twin Cities ETTYftg TJUN 3 0 2014 LIBRA RIES SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGY AT THE MASSACHUSSETTS INSTITUTE OF TECHNOLOGY JUNE 2014 ( Ana B. Oromendia. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publically paper and electronic copies of this thesis document in whole or in part in any medium now know or hereafter created Signature of author: Certified by: Signature redacted I Signature redacted "' Accepted by: Department of Biology June, 2014 I 1 A Angelika Amon Professor of Biology Thesis Supervisor Signature redact d Michael Laub Professor of Biology Chair, Committee for Graduate Students, Microbiology Graduate Program 1 Aneuploidy causes proteotoxic stress in Saccharomyces cerevisiae. By Ana Belen Oromendia Submitted to the Department of Biology on May 1", 2014 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT Gains or losses of entire chromosomes lead to aneuploidy, a condition tolerated poorly in all eukaryotes analyzed to date. How aneuploidy affects organismal and cellular physiology is only beginning to be understood. Aneuploidy also has a profound impact on human health; it is the leading cause of mental retardation and spontaneous abortions and a key characteristic of cancer, as more than 90% of all solid human tumors have aneuploid genomes. Systematic analyses of aneuploid yeast and mouse cells suggested that aneuploidy causes chromosome-specific effects elicited by the amplification of specific genes and general aneuploidy-associated phenotypes Here I describe a phenotype that is shared by most if not all aneuploid yeast cells- I find that aneuploid budding yeast cells are under proteotoxic stress. I show that aneuploid strains are prone to aggregation of endogenous proteins as well as of ectopically expressed hard to fold proteins such as polyQ stretchcontaining proteins. Prion conversion rates are also increased in most aneuploid yeast strains. Protein aggregate formation in aneuploid yeast strains is likely due to limiting protein quality control systems, since I present data showing that at least one chaperone family, Hsp90, is compromised in many aneuploid strains. The link between aneuploidy and the formation and persistence of protein aggregates has important implications for diseases such as cancer and neurodegeneration. Thesis Supervisor: Angelika Amon Title: Professor of Biology 2 This thesis is dedicated with much love and admiration to Ana Maria Vigliocco. "Ifyou are not lost you are at a place that someone else has aheady found..." Junot Diaz 3 Acknowledgements During the course of this thesis, many people have been invaluable in their support, advice and encouragement. First and foremost, I would like to thank my advisor Angelika Amon. Angelika: it has been a privilege to learn how to think about science from you- I couldn't have asked for a better scientific role model. The Amon Lab was an amazing place to learn how to be a scientist and I will take away with me many memories forged in the old CCR and the KI building. Luke, Elcin, Matt, Leon, Folkert and Stefano were an invaluable source of knowledge and technical expertise and always willing to discuss data when I needed a sounding board. I would especially like to thank Jeremy and Michelle for teaching me, and Stacie, Megan and Juliann for teaching me how to teach. Sarah was ever so patient in helping me learn how to work with mammalian cells and scientific discussions with her are some of my greatest memories. I'm thankful for the many fun times and wonderful friendships I forged with Michelle, Sarah, Kristin, Stacie, Megan, Elcin, Luke Matt and Jeremy. A huge thank you goes to my committee members: Frank Solomon and Susan Lindquist. Your input and support was greatly appreciated. I would also like to thank Randy King for participating in my defense. I am incredibly grateful to David Schauer and Alan Grossman for starting the Interdepartmental Microbiology Graduate Program @ MIT and for including me in the founding class. Their dedication to the program, and to my success while at MIT had no bounds. I would especially like to thank Frank Solomon and Alan Grossman for their continuous encouragement- thank you for being straightforward and honest and for believing in me every step of the way; I cannot explain how much it has meant to me. A very special thank you goes to the Massachusetts General Hospital and the wonderful doctors and nurses there, in particular Dr. Christopher Oglivy and Dr. Patricia Musolino. I truly could not have done this without you. The support I have received from many friends during the last years both in the form of lengthy conversations and late night drinks has been invaluable. I am better for having had you in my life. Heather, Marina, Caro, Meche, Jordan and Cristian- thank you for being my family in Boston. I would especially like to thank the MIT Micro dudes:Ben and Tyler, who have been here with me from the very beginning. Saydi, there are no words for how much your love and support has buoyed me through, thank you for always being my #1 cheerleader! Finally, I would like to thank my family. You, and your unrelenting support and love that has no bounds means more than I can ever explain. I thank you for encouraging the curiosity and creativity that has led me to pursue science. To my siblings Mercedes, Clara, Milagros and Manuel- you are my best friends, and your unwavering encouragement has kept me going all these years. To my mom and dad: I am so incredibly grateful for all the sacrifices you have made to give us choices; thank you for always being on my side and encouraging my dreams. To Ana V: you inspire me as a scientist and as a person- I hope that when I grow up I can be half the person you are. 4 Table of contents ABSTRACT 2 ACKNOWLEDGEMENTS 4 TABLE OF CONTENTS 5 CHAPTER 1: 7 ANEUPLOIDY DISRUPTS CELLULAR BALANCE 8 Genome maintenance Comparison between aneuploidy and polyploidy Origins of whole-chromosome aneuploidy Saccharomyces cerevisiaemodels of aneuploidy Cellular consequences of aneuploidy Aneuploidy results in reduced proliferation Transcriptional response to aneuploidy Aneuploidy results in proteome alterations PROTEIN QUALITY CONTROL MAINTAINS THE PROTEOME Protein Folding Controlling Protein Aggregation Protein Degradation Cellular responses to acute proteotoxic stressors ANEUPLOIDY, PROTEIN QUALITY CONTROL AND DISEASE 8 9 9 14 19 21 22 23 29 29 35 36 37 38 Aneuploidy in Cancer Whole-organism aneuploidy Aneuploidy and Neurodegeneration 39 40 43 Aneuploidy and aging 43 Concluding Remarks 44 References 46 CHAPTER 2: 50 Introduction 51 Results 53 Disomic yeast strains harbor a higher load of endogenous protein aggregates. 53 Adaptation to proteotoxic stress is delayed in disomic yeast strains. 57 Meiotic and mitotic chromosome mis-segregation leads to protein aggregate formation. 68 Aneuploid strains fail to efficiently fold the protein quality control sensor VHL. Loss of UBP6 reduces aggregate burden in disomic yeast strains. 71 75 5 76 Hsp90 folding capacity is reduced in many disomic yeast strains. Aneuploid strains are more susceptible to protein aggregates associated with human disease. 80 Discussion Why are aneuploid cells aggregate-prone? Aneuploidy in cancer and neurodegenerative diseases. 87 88 90 Materials and Methods 91 Strains used in this study. All straisn are of the W303 background 97 References 107 CHAPTER 3: 111 Summary of key conclusions 112 Aneuploidy exhausts the cell's protein quality control capacity Why are aneuploid cells aggregate-prone? The folding capacity of chaperones is altered by genomic imbalances Aneuploidy is a chronic stress, distinct form environmental proteotoxic stressors 114 114 118 119 The composition of protein aggregates in aneuploid yeast 122 Aneuploidy in mammalian cells alters protein quality control 127 Interface between aneuploidy, aging and neurodegeneration 129 References 132 6 Chapter 1: Introduction Sections of this introduction have been reproduced with permission from DMM Oromendia, A and Amon, A 'A neuploidy: implicationsfor protein homeostasis and disease' DMM, in press 2013 7 Homeostasis is at the crux of biology. Cells must maintain their karyotipic integrity and, at the same time, ensure the maintenance of their proteome even when faced by stressful growth conditions. I have found that the disruption of a balanced karyotype, i.e. aneuploidy results in a disruption in protein homeostasis. This Introduction will expand first on the consequences of aneuploidy, then on the cellular mechanisms that maintain protein homeostasis and finally explore the interactions they share in the context of human disease. ANEUPLOIDY DISRUPTS CELLULAR BALANCE Genome maintenance The maintenance of stable karyotype, i.e. number and identity of chromosomes, is essential to the success of all species. Species exist with varying chromosomal copies, from haploid (1 copy of each chromosome) to the most common diploid (2 copies) but some plant species can have up to 12 copies of each chromosome. Regardless of ploidy, all organisms carry an equal number of each chromosome ensuring a balanced genome in which genes encoded on different chromosomes are present in the same number of copies. It is this balance that gets disrupted in aneuploid cells. Aneuploidy, defined as a karyotype that is not a whole multiple of the genomic complement results in an 'unbalanced' genome in which chromosomes(s), or pieces of chromosome(s) are missing or supernumerary and thus genes present on different chromosomes are present in varying copy numbers. Several studies have now shown that gene copy number is well correlated with gene expression and, for the most part, well correlated with protein abundance- an imbalance in copy number results in an imbalance of gene products that aneuploid cells are burdened with. Aneuploidy is generally not well 8 tolerated in nature, giving rise to developmental abnormalities of aneuploid organisms and the impaired fitness of aneuploid cells in all species studied to date (reviewed in (Williams and Amon 2009, Torres, 2008). Comparison between aneuploidy and polyploidy Whereas aneuploidy results in an unbalanced, abnormal number of chromosomes and is poorly tolerated in nature, polyploidy does not. Polyploidy is a condition in which cells contain a non-cognate, but balanced number of chromosomes- i.e. cells that of a species that normally maintains a 2n karyotype being tetraploid (4n). Since the relative ratio between gene products is maintained, there is no imbalance for the cell to contend with. Polyploidy, to a degree, is well-tolerated and there are many well documented cases of cells intentionally becoming polyploid to perform their function, such as human megakaryocytes and Drosophila melanogaster salivary gland cells (Lacroix and Maddox 2012). It is clear that while there is an optimal karyotype that each species has evolved to have, modifications that alter chromosome number but maintain genomic balance are far less detrimental than those that generate genomic imbalanceby altering the copy number of only a subset of chromosomes. Origins of whole-chromosome aneuploidy During the course of cell division cells must replicate their DNA and then segregate it equally so that each daughter cell maintains the same chromosomal content as the mother cell. The cell employs a number of mechanisms to ensure that chromosome segregation has occurred before cell division concludes. The process of chromosome segregation begins when the replicated sister chromatids are linked via cohesin molecules. During prophase, 9 each pair of sister chromatids forms attachments to the mitotic spindle so that each chromatid's kinetochore is attached to opposing spindle poles via microtubules. In metaphase, sister chromatids are attached to opposing spindle poles and under tension from pulling forces of rnicrotubules and cohesin molecules holding them together; they are said to be bi-oriented. For accurate chromosome segregation, it is essential to prevent cell cycle progression until all sister chromatid pairs are bi-oriented. The Spindle Assembly Checkpoint (SAC) monitors chromatid attachment and tension and halts the cell cycle until all sister chromatids are properly attached to the mitotic spindle. Once all of the chromatids are appropriately attached, Separase cleaves the cohesin molecules and allows the pulling microtubules to segregate individual chromatids to opposing poles (Figure 1). Figure 1: The Spindle Assembly Checkpoint (SAC) ensures accurate chromosome segregation (a) Cohesion between sister chromatids is retained through metaphase until all attachments to the spindle have been properly made. At the metaphase to anaphase transition, APCCDC20 stimulates the degradation of the inhibitory protein Securin, the degradation of Securin frees Separase to cleave Cohesin. As the chromatids are attached to opposite spindle poles and under tension, they move away from the metaphase plate as the spindle elongates. (b) When chromosomes are not attached, or improperly attached to the spindle, there is a lack of tension. It is this lack of tension, detected, in part, by the kinase Aurora B that activates the Spindle Assembly Checkpoint. MAD2, along with other players, prevents the ubiquitination of Securin by the APC-CDC20. Securin maintains Separase inactive, pausing cell cycle progression. Bypass of the SAC can lead to progression through the cell cycle with improper chromosome attachments resulting in aneuploidy. 10 Figure 1 A Correct attachments B Tension SAC OFF Securi Separase incorrect attachments No tension - SAC ON -' AuroraB MAD2 CDXSecurin _............................. ...... r Separase Compromised SAC function or mis-regulated Separase activity invariably leads to whole-chromosome aneuploidy because the cell cycle is not arrested in cells with unattached or mis-attached chromosomes (Figure 2a). Defects in chromatid cohesion also result in aneuploidy- each chromatid can segregate as it attaches to a microtubule, resulting in almost random chromosome segregation (Figure 2b). Chromatids can also form aberrant kinetochore attachments that are difficult for the SAC to detect. Merotely, when a single sister chromatid kinetochore is attached to microtubules from both spindle poles, is especially difficult to detect as there is still ongoing tension. Often, these resolve by anaphase and do not result in aneuploidy (Thompson and Compton 2008) (Thompson and Compton 2011) but when unequal merotelic attachments occur (kinetochore attached to more microtubules emanating from one pole than from the other), aneuploidy is thought to ensue (Figure 2c) 11 Errors in chromosome segregation in meiosis result in the creation of aneuploid gametes, which can then lead to whole-organism aneuploidy. In mejosis, DNA replication is followed by two rounds of chromosome segregation: first, in Meiosis I homologous chromosomes segregate away from each other, and in Meiosis II sister chromatids segregate. In order to accomplish these orchestrated segregation events, cells have altered the canonical, mitotic chromosome segregation program. To properly segregate homologues, chromosomes undergo crossover events that physically link homologous chromosomes and allow them to align at the Meiosis I metaphase plate, both sister kinetochores must also coorient and attach to the same pole. Additionally, cohesion is lost in a stepwise manner, with arm cohesion being lost first, to allow for homologue segregation in anaphase I and centromere cohesion lost at a later stage to allow for sister chromatid segregation at anaphase II. In metaphase II, sister kinetochores must bi-orient and attach to opposing poles for sister chromatids to segregate to either pole (reviewed in (Miller et al. 2013)). Failure in any of several meiotic chromosome segregation events can lead to mis-segregation, including premature sister chromatid separation, failure to establish crossovers between homologous chromosomes in Meiosis I and various chromosome attachment defects in either Meiosis I or Meiosis II (Figure 2d). Errors in chromosome segregation can arise via many different means, and understanding the consequences of these events on cellular physiology is of critical importance. Aneuploidy has been shown to have severe consequences and to be detrimental in most cases studied to date. 12 FIGURE 2: Whole chromosome aneuploidy arises through errors in mitosis or meiosis (adapted from J. Siegel and Amon 2011) Cells missegregate chromosomes in mitosis by: (a) mutations in the Spindle Assembly Checkpoint (SAC) in which mis-attached kinetochores do not trigger a cell-cycle arrest, (b) premature loss of sister chromatid cohesion where sister chromatids attach to spindle poles and segregate randomly, and (c) merotelic attachments in which a single kinetochore attaches to microtubules emanating from both poles. (e) Aneuploidy can also arise from errors in chromosome segregation in either Meiosis I or Meiosis II. Figure 2 A Spindle Assembly Checkpoint Mutations B Pre-mature Loss of Chromatid Cohesion C Aberrant Kinetochore Attachments [ED E I IC -c *1 D . Meiotic Segregation Errors d mis-segregation durin meiosisi Lurin 0 ' eiosis~ EN [Nx 13 Saccharomyces cerevisiae models of aneuploidy In this thesis I have used aneuploid Saccharomyces cerevisiae strains of various karyotypes generated via three different methods (Figure 3). I generated highly aneuploid, highly genomically unstable strains via triploid meiosis and using mutants that readily missegregate chromosomes during mitosis. Additionally, I used a set of stably aneuploid strains that carry one extra chromosome that were generated by direct chromosome transfer. Using this wide panel of aneuploid strains, I was able to ensure that the phenotypes observed are not due to any particular karyotype nor to the method via which they were constructed; I am confident that the phenotypes observed in the majority of the strains are consequences of being aneuploid. Saccharomyces cerevisiae strains that carry large, random, genomic imbalances were created by inducing missegregation of chromosomes either in meiosis or in mitosis. I created a triploid strain (3n, genotype a/a/a), induced it to undergo meiosis via starvation and recovered the meiotic products (Figure 3a). Triploid cells induced to undergo meiosis produce highly aneuploid progeny, with karyotypes ranging from diploid to highly aneuploid (St Charles et al. 2010). The majority of the aneuploid progeny is inviable (Parry and Cox 1970), but some genetically unstable aneuploid strains can be obtained (Pavelka et al. 2010b) (Sheltzer et al. 2011) (Zhu et al. 2012). As colony formation is a prerequisite for the recovery of these strains, the aneuploidies that cause severe growth defects and do not form colonies will not be analyzed. This approach to generating aneuploid strains is beneficial in that it rapidly allows one to generate a pool of strains with high karyotype variability, as these cells are highly unstable, one is limited to colony formation or single cell assays and must take into account that the analysis will be biased towards 'healthier' aneuploidies that do not impinge greatly on colony formation or growth. 14 Figure 3: Generating aneuploid Saccharomyces cerevisiae strains (Adapted from (Siegel and Amon 2012)) Triploid strains induced to undergo meiosis produce highly aneuploid progeny (a). Using the abortive matings of the karyogamy defective karl,15 strain, aneuploid strains can be generated by single chromosome transfer and selection using markers placed at the same locus on both chromosomes (b). Mitotic chromosome mis-seggregation can be induced by shifting strains carrying temperature sensitive alleles of Iptl or Ndc1O (c) Figure 3 B k; C 1 CN0i 15 N [A 25 "C selection~ N { marker 1 selecion marker 2 _ Seieotionr 1 & 2 15 One can also generate random aneuploidies by inducing chromosome missegregation during mitosis (Figure 3c). Strains harboring temperature-sensitive alleles of genes encoding the kinetochore component Ndc10 or the SAC component Aurora B kinase, Ipli can be arrested in G1 under permissive growth conditions, and induced to mis-segregate chromosomes by shifting them to semi-permissive growth conditions. This treatment results in dramatic chromosome mis-segregation, with 29-35% of cells being unable to correctly segregate a chromosome that is marked by integrating a tandem array of tetO sequences. As these strains also carry a TetR-green fluorescent protein (GFP) fusion, one can visualize the tetR arrays and by extension, track chromosome segregation (GFP-dots) (Oromendia et al. 2012). As with aneuploid strains generated by meiotic chromosome mis-segregation, these strains are highly unstable and are best employed for single cell assays or genetic synthetic interaction analysis with other mutant strains. In order to more carefully characterize aneuploidy and perform population based assays, our lab developed a set of haploid yeast that carry an extra copy of one additional chromosome ((Torres et al. 2007), Figure 3b); these strains have an n+1 karyotype and will be referred to as disomes in this thesis. These disomic yeast strains with defined karyotypes were generated via chromosome transfer from a donor cell to a recipient cell (Figure 4). Disomic strains are low-complexity aneuploidies (only carrying one supernumerary chromosome) but, by adding selectable markers at the same locus in both copies of the disomic chromosome, one can use double selection methods to ensure a stably propagating, pure population of an aneuploid strain with a defined karyotype. These strains have proven to be invaluable in understanding the effects of aneuploidy on cellular physiology, but due to the method in which they are generated one can only create low-complexity (one or two extra chromosomes) aneuploidies. 16 To comprehensively study the effects of aneuploidy on cellular physiology, I have generated aneuploid strains in various different manners. I used strains that carry stable, lowcomplexity aneuploidies and unstable high-complexity aneuploid strains, strains resulting from mitotic or mitotic chromosome mis-segregation and strains that can be maintained as aneuploid via selection. Using this wide panel of aneuploidies I hope to elucidate the general consequences that aneuploidy has on a cell. Figure 4: Generating aneuploid strains via failed karyogamy matings (Adapted from Torres, et al 2007) Strains carrying extra chromosome were generated by a chromosome transfer strategy described by Hugerat et al. (Hugerat and Simchen 1993) A HIS3 cassette is integrated at a particular location on each chromosome using the PCR-based method described by Longetine et al. (Longtine et al. 1998) The strain is then mated to a strain carrying the karlA15 allele, which renders the strain defective in karyogamy (STEP 1).b In addition the strain carries the cyb2-Q37E allele, which confers resistance to cycloheximide in a recessive manner. The mating mixture was then plated on medium lacking histidine and containing 3pg/ml cycloheximide to select for the marked chromosome and to select against diploids and heterokaryons. karz1l5 cells carrying the HIS3 marked chromosome were then mated to cells that carried the kanMX6 cassette at the same genomic locus where the HIS3 was integrated (STEP 2). This strain also carries the cani-100 allele, which confers resistance to canavinine in a recessive manner. Matings were performed and the mating mixture was plated on medium containing G418 and lacking histidine to select for the presence of the disome. To select against mating events the medium also contained canavanine. 17 Figure 4 Step I Mata, xxx::HIS3, LYS2, CYCH2, can 1-100 Mato, karlA15, lys2-801, cyh2-Q37E xxx-,HIS3 Select for: CycR and -His xxx:HIS3 Step 2 Mata, xxx::HIS3, LYS2, CYCH2, can 1-100 Mata, kar1A15, lys2-801, cyh2-Q37E xxx::.kanMX6 xxx..HIS3 xxx HIS3 xxx HIS3 xxx katpMX6 Select for: CanR, -His and KanR xxx kanMX6 18 Cellular consequences of aneuploidy Systematic analyses of aneuploid yeast, mouse and human cells and studies on cancer cell lines suggest that aneuploidy causes chromosome-specific effects that are elicited by the increased (or decreased) number of copies of individual genes and/or combinations of a small number of genes present on the aneuploid chromosome (Tang and Amon 2013). Changes in the gene copy number of regulators of gene expression lead to further disruption of cellular function. Surprisingly, recent studies have shown that aneuploidy also causes chromosome-independent effects, which are a not a consequence of any specific gene imbalance but general consequences of harboring an unbalanced karyotype. These phenotypes include a cell cycle delay in G1 (Torres et al. 2007; Stingele et al. 2012b; Thorburn et al. 2013), metabolic alterations (Williams et al. 2008; Pavelka et al. 2010a), genomic instability (Sheltzer et al. 2011; Zhu et al. 2012) and proteotoxicity (Torres et al. 2007; Tang et al. 2011; Oromendia et al. 2012; Stingele et al. 2012b) (Figure 5). Understanding the origins of these phenotypes is important as this could provide insights into how chromosome mis-segregation and the resulting imbalanced karyotype impacts normal cell physiology and disease states. I describe a subset of these phenotypes in more detail below. 19 Figure 5: Observed characteristics of aneuploid cells in yeast (a) and mammalian cells (b). Adapted from (Siegel and Amon 2012). Blue boxes show observed physiological stresses and pink boxes show conditional changes resulting from aneuploidy. Figure 5 A Yeast Insabilenomic ncreased Protein Synthesis Aneuploidy (ProteinMetabolic CProtein Imbalance r Alterations GSow Energy Stress roteotoxic C Stress nvironmental Stres Response R Mammalian CE An )uploidy Metabolic Alterations increased Hsp72 j ,Reactive Oxygin se dAutophag ---- E nergy Stres S pecies ATM Acvatio Lp38 Activatio ~iva~on + 20 A neuploidy results in reducedprolferation Among the most prevalent and key phenotypes of aneuploid cells is their slower proliferation relative to that of euploid cells. First described in fibroblasts derived from individuals with Down's Syndrome (Segal and McCoy 1974), we now know that this phenotype is a general consequence of aneuploidy. Thorough studies in aneuploid Saccharomyces cerevisiae harboring an extra copy of one or two chromosomes (Torres et al. 2007) or derived from triploid meiosis (Pavelka et al. 2010b) and in SchiZosaccharomycespombe aneuploid cells derived from triploid meiosis (Niwa et al. 2006) showed that, irrespective of which chromosomes are present in excess, aneuploidy results in impaired proliferation. Similarly, aneuploid MEFs containing an extra copy of chromosomes 1, 13, 16 or 19 (Williams et al. 2008) exhibit proliferation defects, and MEFs derived from mice carrying a hypomorphic allele of the SAC component BUBR1 show slower proliferation than euploid MEFs at later passages when aneuploidies are allowed to accumulate (Baker et al. 2004). Aneuploid cells obtained by inducing meiotic non-disjunction, MEFs harboring mutations in SAC component Bubi, or mutations that render the checkpoint component Cdc20 non functional also exhibit proliferation defects and are outcompeted by euploid cells in growth assays (Thompson and Compton 2008; Li et al. 2009). The slow growth phenotype of aneuploid cells has been most extensively studied in S. cerevisiae, in which a recent study has found that aneuploidy results in an extended G1 phase and a delay into entry of the cell cycle that correlates well with the size of the supernumerary chromosome (Thorburn et al. 2013). This phenotype is dependent on the proteornic consequences of aneuploidy, as strains carrying chromosome sized human DNA fragments that can be replicated but do not produce any protein do not display a G1 delay. Both cell growth (cell volume accumulation) and entry into the cell cycle appear to be 21 affected. Although most disomic yeast strains show a cell growth defect, there appear to be no gross defects in global protein synthesis as measured by polysome profiling or ['S]methionine incorporation(Thorburn et al. 2013),although the effects of aneuploidy on these processes may be too subtle to detect by these methods. The growth defect does not appear to be due to diminished amino acid pools or reduced translational efficiency. 10 out of 14 disomic strains analyzed showed a delay in cell cycle entry observed as an increase in critical size (the size at which 50% of cells in a population have budded). All of the strains analyzed show delayed accumulation of the G1 cyclin CLN2 mRNA, and it was shown that high levels of CLN2 suppress the increase in critical size. Accumulation of Cln3, another G1 cyclin was also delayed in all disomes analyzed (Thorburn et al. 2013). It is yet unclear how aneuploidy interferes with the accumulation of Cln3 and whether this is a gene specific effect or a general response to aneuploidy. As it has been observed in almost all aneuploid strains, I favor the idea that the G1 delay is a general consequence of aneuploidy. Interestingly, many environmental stresses (including heat stress) have been shown to cause a transient G1 delay- it is possible that proteotoxic stress in aneuploid yeast is contributing to the G1 delay observed. Transcriptionalresponse to aneuploidy Several lines of evidence suggest that cells respond to the aneuploid state. Most aneuploid cells studied to date exhibit a transcriptional signature associated with slow growth and stress (Torres et al. 2007; Sheltzer et al. 2012; Stingele et al. 2012b; Foijer et al. 2013). Recent studies have shown that aneuploidy elicits a transcriptional response reminiscent of the environmental stress response (ESR) in species as divergent as budding and fission yeast, Arabidopsis thaliana, and human and mouse cell lines. The ESR consists of -300 genes that 22 are upregulated and ~600 genes that are downregulated by various exogenous stresses, including heat shock or oxidative stress (Gasch et al. 2000). Most of these genes also vary in expression in response to growth rate; inducing slow proliferation by nutrient limitation mimics the ESR (Regenberg et al. 2006; Brauer et al. 2008). The high correlation between the ESR-like response seen in aneuploid cells and the transcriptional response observed in slowgrowing S cerevisiae strains suggests that the transcriptional response observed in aneuploid cells is, for the most part, due to the slow proliferation observed in aneuploidy (Sheltzer et al. 2012). A neuploidy results in proteome alterations The unbalanced genome caused by aneuploidy has been shown to translate into an unbalanced proteome - that is to say that the changes in gene dosage for the most part result in equivalent changes in protein levels (twice as much DNA results in twice as much protein, Figure 5). Studies of Saccharomyces cerevisiae aneuploid strains show that the abundance of approximately 80 percent of proteins changes in proportion to gene copy number (Pavelka et al. 2010b; Torres et al. 2010). Interestingly, many of the proteins for which this is not true are subunits of multimeric complexes (Torres et al. 2007). Indeed, often times, subunits that are endogenously expressed in excess because of aneuploidy retain stoichiometric numbers within multimeric complexes (Torres et al. 2007). Stingele and colleagues showed that this is also true in human aneuploid cells {Stingele, 2012 #1121; Torres et al. 2010). Analysis of the transcriptome and proteome of aneuploid human cells generated by chromosome transfer showed that most genes are expressed according to their copy number, and proteins are translated in strong correlation with the abundance of mRNA, resulting in a dramatic change in cellular protein composition (Stingele, 2012 a). 23 Figure 6: DNA, mRNA and protein levels in yeast disomic for chromosome V (Data from Torres et al 2007). Disomic S. cerevisiae strains carry an active, replicating chromosome in one additional copy as evidenced by comparative genome hybridization (CGH, top panel). The extra chromosome is transcribed, as seen by the two-fold increase in mRNA present form that chromosome (microarray, middle panel). The majority of the proteins encoded by the chromosome are also expressed and can be found at close to 2 fold higher levels than those encoded by other chromosomes (SILAC, bottom panel) Figure 6 DISOME V 43- DNA o . 2 (CGH) -2 !hr i Chr V -3 (Aray) 4- -1 3 (RNA -2 shr I Chr V 4. PROTEIN 20 (SILAC) 1t -2 br l ChrV SChromosome Position 24 However, as in aneuploid yeast, human aneuploid cells were also found to maintain a subset of proteins (enriched for complex subunits) at stoichiometric levels even if gene copy number was altered. The regulatory mechanisms responsible for this correcting process have not been elucidated. Overall, these data suggest that, although some proteins are maintained at stoichiometric levels, there is no general whole-chromosome 'gene dosage compensation' mechanism for autosomes in yeast and mammals, as has been observed for sex chromosomes. This might not be the case in all organisms, however. Aneuploid Drosophila S2 cells have been reported to experience dosage compensation at the transcriptional level by means of the male-specific lethal (MSL) complex and general compensation mechanisms that compensate for differences in non-autosomal chromosome copy number (Zhang et al. 2010). Further studies in Drosophilaaneuploid cells are needed to determine the status of their proteome. A key question resulting from the profound effects of aneuploidy on cellular protein composition is whether the simultaneous changes in the relative ratios of many proteins impacts upon the protein quality-control pathways of the cell. Chaperones and the degradation machinery, the 26S proteasome, proteases and autophagy, ensure that all proteins acquire their native conformation and prevent cellular toxicity by reducing the number of aberrant interactions between proteins. In aneuploid cells, these protein qualitycontrol systems must not only attend to the excess proteins produced from additional chromosomes, they must also support all excess subunits of complexes that are not in stoichiometric ratios with their binding partners (Figure 7). 25 Figure 7. Aneuploidy causes proteotoxic stress. (a) Cells use protein quality-control and feedback mechanisms to maintain subunit stoichiometries of complexes whose subunits are encoded by different chromosomes. The protein quality-control (QC) machinery ensures accurate folding and maintains complex subunits that lack a binding partner in a soluble state. Eventually, excess and misfolded subunits must be degraded, as illustrated here by the yellow subunit that has been produced in relative excess. (b) Changes in chromosome number in aneuploid cells (shown here as disomy of the green chromosome) lead to a genomic imbalance that results in stoichiometric protein imbalances. Every subunit encoded by an unbalanced chromosome that functions in a protein complex lacks its binding partner(s) and must rely on cellular chaperones to maintain solubility and, if no binding partner is found, on the cellular proteases for its eventual degradation. This can lead to an increased burden on the protein quality-control systems and the exhaustion of the cellular protein quality-control machinery. 26 Figure 7 A EUPLOID CELLS Chromosomes lA I Complex Subunits B C Protein Complex ABC A X W Chaperone QC B ANEUPLOID CELLS Chromosomes 1 11 111 Complex Subunits A B C Protein Complex ABC V7~ Many protein complex subunits are unstable unless bound to their partners, and will often bind to cellular chaperones to remain soluble until they have formed the complex (Boulon et al. 2010). Several previous studies have indeed hinted to the fact that aneuploidy impacts protein quality-control systems. Budding yeast, mouse and human aneuploid cells exhibit a transcriptional signature that is reminiscent of a stress response and slow growth (Torres et al. 2007; Sheltzer et al. 2012; Stingele et al. 2012a). This transcriptional signature includes upregulation of protein chaperones (Sheltzer et al. 2012). Human aneuploidies generated by chromosome transfer were found to have a transcriptional stress signature that shows up-regulation of lysosome-mediated degradation and p62-dependent autophagy 27 (Stingele et al. 2012a; Stingele et al. 2013). Furthermore, many haploid S. cerevisiae strains harboring an additional chromosome (disomic yeast strains) were found to be sensitive to chemical compounds that impair protein quality control; many disomic yeast strains are sensitive to the proteasome inhibitor MG132, the ribosome poison cycloheximide and the Hsp90 inhibitors radicicol and geldanamycin (Torres et al. 2007). Mouse embryonic fibroblasts (MEFs) trisomic for any of chromosomes 1, 13, 16 or 19 are more sensitive to the Hsp90 inhibitor 17-AAG than are wild-type MEFs (Tang et al. 2011). These results can be interpreted in that that the aneuploid state causes proteotoxic stress leading aneuploid cells to rely more heavily on their protein quality control machinery. Thus, impairing chaperone function via use of chemical chaperone inhibitors is more detrimental to cells that are aneuploid than to cells that carry the appropriate number of chromosomes. This thesis directly tests this possibility. 28 PROTEIN QUALITY CONTROL MAINTAINS THE PROTEOME At the core of cellular biology is the process of converting genetic information into proteins that both carry out the genetic program and provide structural integrity to the cell. The central dogma of molecular biology describes the lifecycle of each individual protein subunit. Protein coding genes are perpetuated in the genome as DNA. When necessary, the DNA is transcribed into mRNA molecules, which are then translated by the ribosome into polypeptides. In order to be functional the majority of polypeptides must acquire a welldefined three-dimensional structure (native structure) and, in many cases, bind to other protein subunits to form a functional protein complex. Once the protein is no longer necessary, it is degraded into individual amino acids that can then be recycled and used in the fabrication of new polypeptides. This process is highly dynamic and energetically costly and at the same time, is affected by almost all external cellular stressors; thus the process of maintaining protein homeostasis (or proteostasis) is one of extreme balance and precision. Protein synthesis is tightly controlled in cells, but in addition, protein folding and protein degradation play an important role in maintaining proteostasis. Protein Folding The information necessary to acquire the native structure is encoded in the primary amino acid sequence and thus, many proteins can fold unassisted in dilute solutions in vitro. In the cellular mileu where the total protein concentration can be as high as 300 mg per ml, acquiring native structure is much more challenging. Inter molecular interations are strongly favored in vivo and since folding intermediates often expose hydrophobic patches, the crowded cellular environment endangers newly synthesized proteins and unstable proteins with high propensity to misfold. Exposed hydrophobic regions constantly pose a threat and 29 non-productive interactions that can result in misfolding and/or aggregation compete with the formation of the native structure. Both protein misfolding and aggregation are detrimental and pose a significant burden to the cell and defects in these processes can result in human disease (Reviewed in (Young et al. 2004; Taipale et al. 2010; Tyedmers et al. 2010). In order to maintain proteostasis and mitigate the effects of heat and other stresses on the proteome, cells have evolved a sophisticated network of protein chaperones. Protein chaperones are intricately involved in the folding and maturation of a protein - from a polypeptide exiting the ribosome acquiring the appropriate three-dimensional structure, to assembly into the appropriate complexes. Molecular chaperone proteins bind to folding intermediates, reducing the conformational space that can be explored and often times preventing aberrant interactions by sequestering hydrophobic patches. Chaperones exist in several structurally unrelated classes and have been classified into families according to their type of enzymatic activity, the co-chaperones they require and the clients that they aid in folding (Hard et al. 2011) and they are named according to their molecular size. Often times, a single polypeptide will interact with different chaperones sequentially, each aiding in a specific aspect of protein folding or complex assembly. It is important to bear in mind that each chaperone family usually has multiple distinct members in each cellular compartment serving to both increase chaperone diversity and ensure redundancy. I will briefly discuss the specifics of the HSP90, HSP70, HSP60 (chaperonins) and small heat shock protein (sHSPs) families here (Figure 8). 30 Figure 8: Molecular Chaperone Mechanisms (adapted from (Richter et al. 2010)) Chaperone model: In general, proteins fold via increasingly structured intermediates (L, L) from the unfolded state (U) to the folded state (N). Protein chaperones bind proteins in nonnative conformations. The shift from the high-affinity binding state to the low-affinity release state is often triggered by ATP binding and hydrolysis. Hsp60/GroE: The GroE machinery consists of two identical rings that enclose a central cavity each. Nonnative protein is bound by the apical domains of the rings, and upon binding of ATP and the cochaperone GroES (caps), the protein is encapsulated and released into the cavity. ATP hydrolysis in one ring results in the release of GroES and substrate protein from the opposite ring. During encapsulation the protein may fold partially or completely. Hsp70: The Hsp70 system comprises two cochaperones, an activating protein (Hsp40/J-protein) and a nucleotide exchange factor (NEF). The activating protein can bind the nonnative protein and deliver it to Hsp70 forming a complex and stimulating its ATPase. The NEF will induce the exchange of nucleotide accelerating the ATPase cycle and the client protein is released Hsp90: In this chaperone system a large number of proteins work together. Often, Hsp70 delivers the substrates to Hsp90. Cochaperones (shown here in purple and yellow) modulate the system (shown here in purple and yellow). ClpB/Hsp104: This chaperone is able to dissolve aggregates by actively pulling proteins through a central channel of the hexameric structure. Refolding occurs upon release, and, to some extent, it can also occur in cooperation with other chaperones. sHsps: sHps are oligomeric complexes that are often activated, by heat or modifications. Many are believed to dissociate into smaller oligomers to become active. sHsps can bind many nonnative proteins per complex. Release requires cooperation with other ATP-dependent chaperones such as Hsp70. 31 Figure 8 chaperone model GroES/HspGQ Hsp7O ClpBIHsplO4 sHsp *rr lowE "Wegh% affnitaLnt AW~PI Hsp9O Hsp9O is at the center of maintaining proteostasis, forming a hub that controls many important signaling pathways (Figure 8, reviewed in (Tfaipale et al. 2010)). Hsp9O functions downstream of Hsp7O binding partially folded polypeptides and cooperating with many cochaperones and regulatory subunits to ensure structural maturation. The activity of Hsp9O is ATP-dependent and closely coordinated with environmental perturbations. Hsp9O is poised to be a buffering force in protein quality control- under normal growth conditions, as it is present in vast excess and can be reduced to 10% of its natural abundance without detrimental consequences to the cell (McClellan et al. 2007; Franzosa et al. 2011). Hsp9O client lists have been notoriously hard to define, perhaps because Hsp9O's essential folding roles seem to be in folding proteins that are central hubs of cellular processes such as 32 regulatory subunits of signal transduction cascades or kinases. Recent genome wide studies have implicated Hsp90 in almost every cellular process from protein trafficking, secretion, RNA processing, signal transduction to telomere maintenance and immunity. The constitutive and inducible forms of Hsp70 are core players in protein quality control (Figure 8, reviewed in (Richter et al. 2010)). Hsp70 (DnaK in E. coli) functions in concert with Hsp40 (DnaJ in E. coli) and nucleotide exchange factors to, in an ATPdependent manner, aid in folding of nascent polypeptides and bind and release partially folded substrates. Hsp70 binds to substrates via small stretches of hydrophobic amino acids, exchanging rapidly in an ATP bound state and binding stably to substrates and Hsp40 after ATP hydrolysis. Rapid cycles of binding and release restrict the conformational folding space that the polypeptide is able to explore and allow for the rapid burial of hydrophobic patches that can partake in aberrant interactions and form aggregates. If the protein is not folded after interaction with the Hsp70/Hsp4O system it may be transferred to the specialized compartment of chaperonins to continue its folding trajectory. Chaperonins are large cage-like ring complexes that function by enclosing the folding polypeptide (up to 60 kDa in size) and isolating it from all other proteins in the cell (reviewed in Hard and Hayer-Hartl 2011). Group I chaperonins, GroEL in bacteria, Hsp60 in eukaryotes are two component systems, with the barrel of the cage formed by the chaperonin and the lid being formed by GroES, in bacteria, or Hsp1O in the case of eukaryotes TRiC/CCT (Figure 8, reviewed in (Dunn et al. 2001)). Group II chaperonins, the system in eukaryotes function under the same premise, but instead of cooperating with another subunit to form a closed cage, they undergo conformational changes to enclose the structure. Chaperonins function in an ATP dependent manner, coordinating the encapsulation of the substrate with hydrolysis of the ATP molecule. The 33 encapsulated protein is free to fold in the chaperonin enclosure until it is released (10s in the GroEL/ES system, longer in the TrIC/CCT complex). Still unfolded substrates can re-bind and the process can be repeated until the protein has acquired its native fold or it is transferred to a different chaperone. Although chaperonins do not actively assist in folding, they have been shown to dramatically accelerate the speed of folding, probably by spatial confinement and the prevention of aberrant interactions and aggregation with other proteins. In S. cerevisiae the TrIC/CCT complex is essential for the folding of a small subset of proteins, but within these are proteins of high abundance and extreme importance in structural integrity of the cell such as actin and tubulin. Small heat shock proteins (Figure 8, reviewed in (Richter et al. 2010)) are not as cohesive of a protein family as HSP90 or HSP70 are. sHSPs are usually monomeric proteins that bind to hydrophobic patches of amino acids. For the most part, their clients have not been well defined, but they are thought to be unstable folding intermediates and that the binding of sHSPs prevents aberrant interactions. sHSPs are thought to play a role in protein complex formation, binding to one protein subunit and occluding the binding interface (usually highly hydrophobic) until the binding partner is found and the complex is formed. There is a vast network of proteins whose function is to ensure protein folding within the cell. Protein chaperones are both diverse and specialized, and while some assist in general folding of proteins, many have a defined subset of protein clients whose folding they aid. As protein homeostasis is a process of utmost importance, protein chaperones also maintain a large amount of redundancy, with many having obligate clients but being able to assist in folding of others if necessary. To cope with severe folding stress- many chaperones have two variants, one that is constitutively expressed at low levels and another whose expression is induced by proteotoxicity. In summary, the cell has developed a robust system 34 of protein folding factors to minimize aberrant interactions between proteins and ensure peptides acquire the appropriate 3-dimensional structure. Controlling Protein Aggregation When polypeptides cannot fold into their native structure and remain misfolded, if they partially unfold after being properly folded or if they are terminally damaged by oxidation or carbonylation, they become aggregate-prone. Assembly defects in protein complexes, as would happen when a required subunit is not expressed, can also lead to aggregation of the existing subunits as hydrophobic patches that would be buried within the complex remain exposed and form aberrant interactions. In addition to folding assistance provided by chaperones, the cell utilizes chaperones to solubilize protein aggregates and utilizes diverse mechanisms to prevent toxicity from aggregated proteins. The Hsp10O chaperone family is comprised by members of the AAA ATPases, most notably ClpB in bacteria and Hsp104 in yeast. Both ClpB and Hsp104 have disaggregating capabilities, using ATP hydrolysis to break apart protein aggregates (Figure 8). The mechanism by which they do this is unclear, but they are thought to thread the misfolded proteins through a central pore of their hexameric ring, leaving the client protein in an unfolded state so that it can refold either on its own or assisted by the Hsp70/Hsp4O machinery (Richter et al. 2010, Mogk, 2004, Tyedmers, 2010). Although no homologues of Hsp104 have been found in higher eukaryotes, disaggregation activity has been attributed to the mammalian chaperone system comprised of Hsp110 and Hsp70/Hsp40 (Shorter 2011). Yeast cells also sequester certain types of protein aggregates, usually those that cannot be refolded, in special compartments The JUNQ (juxtanuclear quality control compartment) transiently accumulates aggregated proteins that are ubiquitinated and destined for 35 degradation whereas the IPOD (Insoluble protein deposit) houses insoluble terminatally aggregated proteins such as polyQ or carbonylated proteins(Kaganovich et al. 2008). When a cell is unable to disaggregate and refold aggregated proteins, degradation of the aggregated proteins is a viable alternative to alleviate toxicity. Protein Degradation In order to cope with alterations in protein homeostasis, cells degrade excess, misfolded and aggregated protein subunits and aberrant peptides by means of the Ubiquitin Proteasome System (UPS) or via autophagy. The 26S proteasome is the central macromolecular machine responsible for the degradation of proteins and protein aggregates. Its functions are so essential to the cell that partially inhibiting its function can lead to neurodegeneration and complete inhibition is lethal (Bedford et al. 2008). The 26S proteasome is comprised of a core, barrel-like particle (20S subunit) and two regulatory complexes (19S) that function as lids. Proteins are recognized and targeted for degradation by E3 ubiquitin ligases that attach ubiquitin moieties. Specialized proteins that contain UBL (ubiquitin like) and UBA (ubiquitin associated) domains act as adaptors between the target protein (the ubiquitin moieties are bound by the UBA domain) and the 19S cap (binds the UBL domains). The proteins targeted for degradation are deubiquitinated, unfolded and threaded through the core particle. The recognition and binding of a substrate to the 19S cap is an ATP dependent process, and the ATP molecule is required for unfolding, but not translocation into the pore. Proteolysis occurs in the core particle through a threonine-dependent nucleophilic attack and results in short stretches of amino acids that can then be further processed by cytosolic proteases and recycled into new polypeptides. 36 Protein degradation is mediated not only by the proteasome but cells can additionally deploy autophagy as a means of protein quality control (Kubota 2009). Misfolded proteins are sequestered into aggregates and, in a p62-dependent manner, are targeted for autophagy. Autophagy utilizes double-membraned structures that engulph the cytosolic target proteins forming an autophagosome which then fuses with the lysosome for degradation of their content (Bukau et al 2010). A key player in autophagosome formation is the membrane protein LC3/Atg8; upon autophagy induction, LC3 is conjugated to phosphatidylethanolamine and recruited to the membranes of the nascent autophagosome. One of the many ways one can monitor autophagy is by assessing the number of LC3 foci, or by assaying the abundance of LC3-II, the autophagosome-specific, lipidated form of LC3. Cellular responses to acute proteotoxic stressors In order to maintain protein homeostasis under acute insults to proteostasis, there are transcriptional programs that cells implement when faced with abnormal quantities of misfolded proteins. These transcriptional programs are distinct according to which cellular compartment is being assaulted by protein misfolding but they are all transient, tailored to temporary stressors. The main goal of these programs is to reduce the folding burden (by reducing the number of polypeptides being produced) and to enhance the cell's folding capacity (by increasing the number of protein chaperones). The best studied is the program elicited by the general misfolding of cytosolic proteins elicited by exposure to high temperature and thus named the 'heat hock response' (HSR). High temperatures result in general protein misfolding which leads to the activation of the transcription factor HSF1 (heat shock factor 1) that then results in the up-regulation of a subset of genes enriched for protein chaperones and the down-regulation of genes involved in protein synthesis 37 (reviewed in (Richter et al. 2010)). Hsfl is kept in an inactive complex together with components of the Hsp90 chaperone system. In a state of heat shock, the high abundance of misfolded proteins is thought to titrate away the chaperones bound to Hsfl. In complex with chaperone, Hsf1 is found as a monomer but its release leads to homotrimerization and transport into the nucleus. There, Hsf1 is hyperphosphorylated by several kinases (Holmberg et al. 2001). Further modification events, like sumoylation, regulate the activity of the final transcription factor complex (Hietakangas et al. 2003). Complex regulatory feedback ensures that the response is transient so as to return to normal levels of protein production and chaperone abundance once the proteotoxic stress has been relieved. Misfolded proteins in the endoplasmic reticulum (ER) result in a similar, but distinct response termed the Unfolded Protein Response (UPR). The UPR is also transient, and results in the up-regulation of ER specific chaperones and a general, temporary, reduction in protein synthesis (Walter and Ron 2011). Studies in mammalian cells have also recently described the mitoUPR (Mitochondria Unfolded Protein Response). Details are far less clear, but the essence of the response is the same: misfolded proteins in the mitochondria result in a signal that translates to a temporary decrease in protein production and an increase in protein quality control capacity (Haynes and Ron 2010). In summary, there are well- understood transcriptional programs that aid in coping with abrupt changes in misfolded proteins caused by disruptions of protein homeostasis. ANEUPLOIDY, PROTEIN QUALITY CONTROL AND DISEASE The connection between aneuploidy and disease has been at the forefront of the study of aneuploidy. David van Hansemann first described unbalanced mitoses in 1890. Theodor Boveri (1912) expanded upon his early description of aneuploid sea urchin 38 embryos to postulate that aneuploid cells could result in tumor formation. Aneuploidy of chromosome 21 was described as the cause of Down's syndrome by Lejeune in 1959 (Lejeune et al. 1959). Recent studies have described associations between the aneuploid state and neurodegenerative diseases and aging. Here I expand upon the most common conditions associated with aneuploidy. Aneuploidy in Cancer Aneuploidy is extremely prevalent in solid tumors, with 7 0- 9 0% estimated to have an unbalanced karyotype (Weaver and Cleveland 2006; Duijf and Benezra 2013). Cancer cells have also long been considered 'chaperone addicted' (Neckers 2002) and Hsp90 inhibitors are currently being developed as chemotherapeutics (Wagner et al. 2013). The dependency of tumors on chaperones has been attributed to the need to efficiently fold oncogene products, which are often kinases and thus Hsp90 clients. However, the high levels of aneuploidy in cancer cells, and the proteotoxic stress that stems from such aneuploidy, could provide an additional explanation for their chaperone addiction. Further investigation of compounds that increase chaperone burden or that inhibit the function of chaperones might lead to the discovery of new cancer therapeutics with efficacy in a broad spectrum of human tumors. The high degree of aneuploidy observed in cancers also begs the question of whether cancer cells have evolved mechanisms that allow them to tolerate high levels of karyotypic imbalances. One aneuploidy-tolerating mutation appears to be loss of p53 function. In normal cells, chromosome mis-segregation leads to activation of the tumor suppressor p5 3 ; the mechanisms whereby this occurs are still being elucidated and might be caused by multiple aspects of chromosome mis-segregation (Pavelka et al. 2010a; Thompson and Compton 2010; Janssen et al. 2011). Generating a comprehensive list of genetic alterations 39 that ameliorate the effects of aneuploidy and their characterization will shed light on tumor evolution. It will allow us to address important questions such as when such mutations arise with respect to aneuploidy and whether and how they contribute to tumorigenesis. Compounds that neutralize aneuploidy-tolerating mutations could also provide new avenues of cancer treatment. Whole-organism aneuploidy In addition to cancer, autosomal aneuploidy has been associated with numerous human conditions that result in impaired development. In humans, three viable trisomies have been described. An additional copy of chromosome 21 leads to Down syndrome, chromosome 18 to Edward's syndrome and a trisomy of chromosome 13 to Patau syndrome. Of these, only Down Syndrome individuals survive past childhood. It will be interesting to determine whether protein quality-control systems are affected in individuals with these constitutional aneuploidies. Chromosome 21 harbors the fewest genes of all human chromosomes and might thus not cause a significant burden on the cellular protein qualitycontrol pathways. Determining the contribution of impaired protein homeostasis to the pleiotropic phenotypes of this syndrome could nevertheless be warranted because Down syndrome is strongly associated with a protein-folding disease. Individuals with Down syndrome are predisposed to early-onset Alzheimer's Disease (AD). Although the main cause of AD in Down syndrome individuals is likely to be the additional copy of the APP gene encoded by chromosome 21 (reviewed in (Kingsbury et al. 2006), mice overexpressing APP (which encodes amyloid beta A4 protein) do not fully recapitulate all the Alzheimer'slike phenotypes seen in Down syndrome mouse models (Cataldo et al. 2003). Conversely, mouse models of Down syndrome that lack the APP gene still exhibit some of the 40 Alzheimer's-like pathologies (Table 1), suggesting that duplication of the A PP gene may not be the only cause of early-onset Alzheimer's disease in Down syndrome individuals. Thus, perhaps a reduced ability to maintain protein homeostasis contributes to the Alzheimer's disease pathology in individuals with Down syndrome. Table 1. Comparison of the phenotypes associated with transgenic mouse models of Down's syndrome or Alzheimer's disease The two mouse models of Down syndrome are Ts65Dn and TslCje. Ts65Dn mice are trisomic for the distal region of chromosome 16 (92 genes homologous to human chromosome 21 from APP to MXJ); this segment contains nearly two-thirds of the human chromosome 21 homologous genes, including the Down syndrome critical region (DSCR) and the APP gene. Ts65Dn mice are also trisomic for a segment of mouse chromosome 17 (60 genes) that is non-homologous to genes on human chromosome 21. TslCje mice are trisomic for a smaller region of chromosome 16 that includes the DSCR but not APP (67 genes homologous to chromosome 21, from SODI to MX1, approximately two-thirds of the trisomic region of Ts65Dn mice), and they are monosomic for the telomeric region of mouse Chr 12 (seven genes) (Cataldo et al. 2003). In addition to these two mouse models of Down syndrome, transgenic mice have been generated that harbor an additional copy of a mutant form of APP (K670M/N671L) that has been identified in a Swedish family with early-onset AD ('APP overexpression', Table 1). Although many of the phenotypes are shared between the mice, an increased copy of APP is not sufficient to recapitulate all of the Alzheimer-related phenotypes of Down's syndrome mouse models. TABLE 1 41 Phenotype APP status Down Syndrome Alzheimer's disease (APP Ts65Dn TslCjc overexpression) 3 genomic 2 genomic copies High levels of mutant APP copies Cognitive abnormalities YES YES YES Age-related atrophy and YES NO YES YES NO NO NO NO YES degeneration of cholinergic neurons Age-related endosomal pathologies Extracellular $-amyloid aggregates In addition to the constitutive aneuploidies of chromosomes 13, 18 and 21, mutations in genes encoding the spindle assembly checkpoint component BUBR1 or centrosome components have been shown to lead to mosaic variegated aneuploidy (MVA), a disease characterized by aneuploidies showing a random widespread distribution in the body (Hanks et al. 2004; Snape et al. 2011). There are no published evaluations of proteotoxicity in MVA cell lines, but, given that protein quality-control systems have also been shown to be impaired in complexly aneuploid yeast strains (haploid strains that are aneuploid for more than one chromosome) (Oromendia et al. 2012), it would be of interest to investigate whether the same is true in the case of MVA patients and to determine how this contributes to the disease phenotype. 42 Aneuploidy and Neurodegeneration Finally, neurodegenerative diseases are protein-folding diseases. Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia and Huntington's disease are all characterized by the misfolding and aggregation of specific proteins. Intriguingly, aneuploid yeast strains were found to be more prone than wild type strains to form aggregates of a hard-to-fold protein containing a polyQ stretch, which is also considered a model for Huntington's disease. Expressing this polyQ protein also impairs proliferation of aneuploid yeast strains more than that of euploid controls, indicating that expression of a hard-to-fold protein affects the fitness of aneuploid cells (Oromendia et al. 2012). Could aneuploidy be a contributor to neurodegenerative protein-folding diseases? Several studies have suggested that as many as 30% of embryonic neurons and 15-20% of adult neurons harbor aneuploidies (Rehen et al. 2001; Rehen et al. 2005; Yurov et al. 2005; Yurov et al. 2007). On the other hand, in a recent study that performed single-cell whole genome sequencing of neurons, high levels of copy number variation (CNVs) but no increased aneuploidy was described (McConnell et al. 2013). Why aneuploidy would be more prevalent in neurons compared with cells of other tissues is unclear, but it would provide an intriguing explanation for the prevalence of protein-folding diseases in this cell type. Future studies and additional methods to assess aneuploidy in tissues will be necessary to assess the degree and types of aneuploidy comprehensively in the brain and to determine the effects, if any, of aneuploidy on neurodegenerative diseases. Aneuploidy and aging All organisms age, and this process is characterized by, among other phenotypes, the following: genomic instability, epigenetic alterations, deregulated nutrient sensing, 43 mitochondrial dysfunction and loss of proteostasis (reviewed in (Lopez-Otin et al. 2013). Furthermore, aging is the primary risk factor for major human diseases, including cancer, diabetes, cardiovascular disorders and neurodegenerative pathologies. Interestingly, recent studies by van Deursen and coworkers have provided intriguing links between aneuploidy and the aging process. They found that mice carrying hypomorphic alleles in the spindle assembly checkpoint gene BUBRI, which also serves as a mouse model for MVA, harbor high levels of aneuploidy (Baker et al. 2004). Remarkably, these animals age prematurely. Mice carrying hypomorphic alleles of BUBRI prematurely develop phenotypes characteristic of old age such as cataracts, sarcopenia, growth retardation, muscle wasting, fat loss and cardiac arrhythmias (Baker et al. 2004; Wijshake et al. 2012; Baker et al. 2013a; Baker et al. 2013b). Intriguingly, overexpression of BUBRI has the opposite effects - it leads to a reduction in chromosome mis-segregation and hence aneuploidy (Baker et al. 2013a), and the animals live longer and have a longer life without ailments (health-span) Furthermore, cardiac function is increased, and muscle and renal atrophy and glomerulosclerosis are reduced (Baker et al. 2013). Exactly how aneuploidy might result in aging remains to be determined, but I propose that the systemic impacts of aneuploidy on cell physiology, such as proteotoxicity, as discussed here, together with metabolic changes and genomic instability, are the source of aneuploidy-induced aging. It will be very interesting to determine whether mutations that suppress the adverse effects of aneuploidy also delay aging and extend life and health span. Concluding Remarks Aneuploidy has a profound impact on most, if not all, cellular functions. This thesis is centered on the consequences of aneuploidy on the protein quality control mechanisms of 44 the cell and the implications this could have on our understanding of human diseases and aging. Aneuploidy has been shown to cause proteotoxic stress in yeast and mammalian cells. Proteotoxicity is a consequence of aneuploidy irrespective of the identity of the supernumerary chromosomes, and thus it is a phenotype inherent to the aneuploid state itself. In this thesis, I will describe the consequences that aneuploidy has on protein homeostasis that lead to an increased prevalence of protein aggregates when compared to euploid cells. I have found that, not only does aneuploidy lead to increased endogenous protein aggregates but it precludes the folding of known protein substrates, and it directly affects the folding capacity of at least one chaperone: Hsp90. In impinging protein quality control, an unbalanced karyotype also sensitizes strains to hard to fold disease proteins and renders them more susceptible to prion conversion. The impact aneuploidy has on essential cellular processes such as protein quality control could be exploited as, a new direction for treatment of the many ailments that are connected to aneuploidy, either in cause or in consequence. The sensitivity of aneuploid cells to disruptions in protein quality control could be used to develop therapeutic and treatment protocols that selectively impale cells that have not maintained the original karyotype. Additionally, it is interesting to ponder the thought of enhancing protein quality control abilities of the cell (either via a chemical enhancer or gene therapy) to counter-act the detrimental effects of an unbalanced karyotype in cases of whole-organism aneuploidy such as Downs Syndrome or as a way to ameliorate the steady decline of aging cells. Understanding the full impact of this condition on cells and organisms will not only deepen our knowledge of the consequences of an imbalanced karyotype but will provide fundamental insights into developmental disabilities such as Down syndrome and diseases such as cancer. 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This imbalance is generally not well tolerated in nature, as evidenced by the impaired fitness of aneuploid cells and organisms (reviewed in (Torres et al. 2008; Williams and Amon 2009)). The condition also has a profound impact on human health. Aneuploidy is the leading cause of mental retardation and spontaneous abortions and a key characteristic of cancer, as more than 90% of all solid human tumors harbor aneuploid genomes (Weaver and Cleveland 2006). Systematic analyses of aneuploid yeast and mouse cells suggest that aneuploidy causes chromosome-specific effects that are elicited by the duplication/deletion of individual genes or combinations of a small number of genes present on the aneuploid chromosome (Torres et al. 2007; Pavelka et al. 2010; Torres et al. 2010; Tang et al. 2011). Aneuploid yeast and mammalian cells also share a set of phenotypes, collectively called the aneuploidyassociated stresses (Torres et al. 2007; Williams et al. 2008; Tang et al. 2011), indicating that the aneuploid state per se impacts cell physiology. Aneuploidy impairs proliferation of budding and fission yeast cells as well as of mammalian cells under standard growth conditions (Baker et al. 2004; Niwa et al. 2006; Torres et al. 2007; Thompson and Compton 2008; Williams et al. 2008; Li et al. 2009; Pavelka et al. 2010), with a delay at the G1 - S phase transition being especially prominent (Niwa et al. 2006; Torres et al. 2007; (Stingele et al. 2012). Whole chromosomal aneuploidies also lead to a transcriptional response. A gene expression signature similar to the environmental stress response (ESR; (Gasch et al. 2000a)) in budding yeast has been observed in aneuploid budding and fission yeast strains, A rabidopsis, mouse and human cells (Sheltzer et al. 2012). Lastly, aneuploid cells exhibit phenotypes characteristic of disruption of protein homeoastasis. Aneuploid budding yeast 51 strains and trisomic mouse embryonic fibroblasts show increased sensitivity to compounds that interfere with protein folding and turnover (Torres et al. 2007; Pavelka et al. 2010; Torres et al. 2010; Tang et al. 2011). Understanding the phenotypes shared by many different types of aneuploidies is of particular importance, as this could provide insights into how an unbalanced karyotype impacts normal cellular physiology and disease states such as cancer. Here we investigate the consequences of one of the general effects of aneuploidy disruption of cellular protein homeostasis. Maintaining the proteome is essential for cell survival. Nascent peptides must be folded, proteins that are unfolded have to be refolded, and terminally damaged proteins must be degraded. Additionally, multi-protein complexes must be properly assembled. The cell relies on molecular chaperones to aid in the folding and refolding of proteins and the assembly of multi-protein complexes, as well as on the 26S proteasome and vacuolar proteases to degrade proteins that are terminally misfolded (Tyedmers et al. 2010; Houck et al. 2012). The chaperone and ubiquitin-proteasome systems function in concert to ensure protein homeostasis. When cells experience proteotoxic stress, that is, when protein qualitycontrol pathways such as the chaperone systems and the proteasomal degradation machinery are compromised or overwhelmed, misfolded proteins are not eliminated and aggregates form (Houck et al. 2012). Misfolded proteins not only inflict a fitness cost (Geiler-Samerotte et al. 2011), they are also associated with human disease. Highly-structured aggregates have been linked to neurodegenerative pathologies, including Huntington's, Alzheimer's and Parkinson's diseases, as well as prion diseases such as Kuru and Creutzfeld-Jacob Syndrome (reviewed in (Goedert et al. 2010)). We previously generated 13 budding yeast strains harboring an additional copy of a single yeast chromosome, called disomes. These strains exhibit, among other deleterious 52 phenotypes, increased sensitivity to high temperature, to inhibitors of protein synthesis and folding, as well as chemical and genetic perturbation of proteasomal degradation (Torres et al. 2007; Torres et al. 2010). Furthermore, we found that increasing proteoasomal degradation by deleting the gene encoding the deubiquitinating enzyme Ubp6 improves the proliferative abilities of a subset of disomic yeast strains. Together with the observation that the additional chromosomes are actively transcribed and translated (Torres et al. 2007; Pavelka et al. 2010; Torres et al. 2010), these studies suggest that aneuploidy alters the cell's proteome resulting in proteotoxic stress and implicate the ubiquitin-proteasome pathway in the survival of aneuploid cells. However, direct evidence of proteotoxicity in aneuploid cells, and the role of chaperones in the generation of proteotoxic stress in aneuploid cells has thus far been lacking. Here we show that aneuploid yeast cells are prone to protein aggregate formation. Aneuploid yeast strains generated by a variety of different methods have defects in aggregate clearance and exhibit increased sensitivity to aggregate-prone proteins. The association between aneuploidy and protein aggregation uncovered in this study could have important implications for the pathology and treatment of diseases such as cancer and neurodegeneration, which have both been associated with aneuploidy. Results Disomicyeaststrains harbora higher load of endogenousprotein aggregates. Introduction of whole chromosomes substantially alters the cell's proteome because most genes present on the additional chromosome are expressed according to gene copy number (Torres et al. 2007; Torres et al. 2010). This may impact protein homeostasis mechanisms. To test this possibility we analyzed the subcellular localization of the disaggregase Hsp104. Under standard growth conditions, the Hsp104 chaperone fused to 53 eGFP is diffusely localized throughout the cell, but the protein also co-localizes with protein aggregates, manifesting as Hspl04-eGFP foci (Liu et al. 2010) (Figure 1A, B). All 13 disomic strains analyzed showed a significant increase in the percentage of cells harboring Hsp104eGFP foci compared to the euploid control (Figure 1A, B). Increased aggregate formation was not due to slowed proliferation caused by aneuploidy (Torres et al. 2007) because temperature-sensitive cdc23-1 and cdc28-4 strains grow slowly at the permissive temperature but do not harbor additional aggregates (Figure 1C). Our data further suggest that it is the increased protein load generated from the additional chromosome that leads to increased protein aggregation. We did not observe an increase in the percentage of cells with Hsp104eGFP foci in strains that contain yeast artificial chromosomes (YACs; Figure 1D) that carry human DNA but generate no yeast proteins and very few if any other peptides and proteins (Foote et al. 1989; Torres et al. 2007). As protein aggregates are the consequence of misfolded proteins, our data suggest that aneuploid cells are challenged to fold proteins efficiently and/or to process protein aggregates appropriately. 54 Figure 1: Disonic yeast strains harbor an increased protein aggregate load. (A) Wild-type and disomic yeast strains containing an HSP04-eGFPfusion were grown to exponential phase in YEPD, and the percentage of cells harboring Hspl04-eGFP foci was determined (n=3; SEM, n=100 cells/time point; **P<0.005; ***P<0.0005; Student's t test). Strain order: A31392, A31393, A31394, A31395, A31396, A31397, A31398, A31399, A31400, A31401, A31402, A31403, A31404, A31405. (B) Images of Hspl04-eGFP aggregates. Aggregates are in green, DNA in blue. (C, D) WT (A25654), cdc23-1 (A29766) and cdc284 (A29765) strains (C) and strains harboring YACs containing 580kb (A28922) or 670kb (A28925) of human DNA (D) were grown as in (A) to determine the percentage of cells with Hspl04-eGFP aggregates. (E) Quantification of Hspl04-eGFP foci in trisomic yeast strains grown as in (A). Strains in order: A31406, A31407, A31408, A31409, A31410. Note that the number of cells with Hspl04-eGFP aggregates in diploid cultures is lower than in haploid cultures. The basis for this is at present unclear. (F) Strains grown at 25*C were shifted to 37*C. The percentage of cells with Hspl04-eGFP aggregates was determined at the indicated times after temperature shift (n=100 cells/time point). Two replicas of this experiment are shown in Figure S1. Strains are the same as in (A). 55 Figure 1 B A60- 25'C x~ a.50- T U- I 4030- *** I C) 2010. I- LI z 04 C -$\0 " D F 20- L20- U C.R 10- 0. 10. J-h- 0 0 0 -kV E L U) C6iC6, 10080U60 (9 40 C) 20J 0v 0 10080 60 40 20 0( 0 U ,C150 0 U- iT 0. U) C.) U, Q0 0 <tp \0 00 4$ ~. 0 .00 + U) ~_wr Dis I Dis VIII -N+- Dis IX -Ii- Dis X -S- Dis XI -a- -0- 1 2 3 4 1 -+- Dis 11 -0- -0- Dis XV 1 2 3 4 -- WT Dis IV -0- Dis V --- Dis XI -- u- 6040 20 00 Dis XIII -- Dis XVI 100 -80 ['1 -C - -V 37'C Eu Eu I ['1 25'C -0- Dis XIV 2 3 4 Time (h) 56 We also found that, as with all other aneuploidy-associated phenotypes (Torres et al., 2007), increasing ploidy suppressed aggregate formation. The percentage of cells harboring Hspl04-eGFP foci in diploid strains carrying an additional chromosome (trisomic strains) is significantly lower than that of haploid strains with an extra chromosome (compare Figure 1A and Figure 1E). Many subunits of protein complexes require the assistance of protein chaperones to fold. These proteins then acquire a stable conformation by binding to the complex's other subunits. If one of the components is present in excess and cannot exist stably as an uncomplexed subunit, it requires the continuous assistance of chaperones to prevent aggregation (Tyedmers et al. 2010). As a result, chaperones cannot assist other folding reactions and the general folding capacity of the cell is reduced. The observation that increasing ploidy reduces aggregate formation suggests that the proteotoxic stress in aneuploid cells could, in part, be a result of stoichiometric imbalances caused by the proteins encoded on the unbalanced chromosomes. Decreasing the ratio of uncomplexed proteins to complexed proteins reduces the protein aggregate load of aneuploid yeast. The observation that aggregate formation in many trisomic strains is not elevated compared to diploid controls further suggests that cells either have the ability to compensate for some genomic imbalances and/or that diploids have a higher folding capacity than haploids. A daptation to proteotoxic stress is delayed in disomicyeaststrains. If disomic yeast strains experience increased proteotoxic stress, they may be delayed in responding or adapting to conditions that induce proteotoxicity. To test whether disomic yeast strains are delayed in adapting to proteotoxic stress-inducing growth conditions, we monitored Hspl04-eGFP foci after shift to high temperature (37*C). Virtually all wild-type and disomic cells contained Hspl04-eGFP foci within an hour of temperature shift (Figure 57 1F). However, whereas wild-type cells cleared the aggregates by 4 hours, all disomes except for disomes IV and XIV adapted to heat stress with slower kinetics (Figure 1F, 2). This delayed adaptation to high temperature was not due to an inability to mount a heat-shock response, as judged by microarray analysis of aneuploid cells adapting to thermal stress (Figure 3). Activation of the unfolded protein response (UPR) in the endoplasmic reticulum was also unaffected in aneuploid strains; splicing of the UPR gene HA C1 in the disomes was similar to that in wild-type cells, both under normal conditions and under conditions when the UPR is induced (Figure 4). Figure 2: Behavior of Hspl04-eGFP aggregates in response to heat stress. Two independent repeats of the experiment shown in Figure 1F are shown in (A) and (B). Strains grown at 25*C in YEPD were collected on a filter and shifted to YEPD pre-warmed to 37*C. The percentage of cells with Hspl04-eGFP aggregates was determined at the indicated times after temperature shift (n=100/time point). Strains: A31392, A31393, A31394, A31395, A31396, A31397, A31398, A31399, A31400, A31401, A31402, A31403, A31404, A31405. 58 Figure 2 B A .5 0. a 100- + -o-w-o- 4 80 601 1m2 0 3 Dish Dis XIII Dis XVI Dis XV -- 0-*+ 40 20 U, _ ,__ o 1 0 ,___ ,___ 3 2 Time (h) Time (h) 3 4 3 0wr W7 80 Dis I -o- Dis VIII -*- Dis IX -a- Dis X -.Dis XI 40 0o Dis IV -o- Dis V -N- Dis XII -o- Dis XIV 2 U100- 1 *1 404 20 1 2 Time (h) ,_ 60. o0 20 0 4 -100-+WT D-0 0 T Dis I -o- Dis Vill Dis IX -c Dis X Dis XI 6-N- M 6-o- Ile Time (h) -100 A o 4 + W + Dis|1 Dis XIII -*Dis XVI -o- Dis XV 10080 +WT 2 3 4 Time (h) U100 P 80 60 WT + Dis IV -o- Dis V - Dis XII -o- Dis XIV + 20 o 1 2 3 4 Time (h) 59 Figure 3: The heat shock response is intact in disomic yeast strains. WT and disomic yeast strains were grown at 25*C and shifted to 37*C. RNA samples were taken 0, 5, 15 and 30 minutes after shift. RNA extracted from WT cells grown continuously at 25*C was used as reference for all samples. Data were mined for genes involved in the heat-shock response (Gasch et al. 2000a)(Gasch et al. 2000b), and those present in the extra chromosome were removed from the analysis (grey boxes). Data set was split into those genes that are upregulated and those that are down regulated in the heat-shock response. (A) The expression of genes involved in the heat-shock response in disomic yeast after shifting to 37*C. Yellow shows upregulated genes and blue shows downregulated genes. Shown are the unclustered data after being zero-transformed. (B) The average expression changes of up- (above x-axis) and down- (below x-axis) regulated genes displayed in (A) are shown. 60 Figure 3 A WT o minutes at 37C DisV DisX Dis XIV Dis S 15 30 o s ; is 30 0 s 15 n 0 S 15 n 0 minutes at 37C 3,00 1.00 0.00 -1.00 1-.00 a 4) a a -C 0 'M S! a M3 a5 CO 0. B 2- I 1p 10 -, 0i> -1 20 minutes at 370C 30 -.- - Dis. V Dis X Dis XIV Dis XVI -3- 61 Figure 4: The Unfolded Protein Response (UPR) in disomic yeast strains. Wild type and disomic strains were grown at 25*C. The culture was split and one half was treated with 2pg/ml of Tunicamycin (TM), a compound known to induce the UPR. Samples were collected 2 hours later and the amount of unspliced (upper band) and spliced (lower band) HA C1 was determined. The red bar indicates the location of the probe. Strains in order are: WT (A22361), Dis I (A6863), Dis II (A6865), Dis VIII (A27036), Dis X (A21986), Dis XI (A28266) Dis XIII (A21987) and Dis XIV (A28344). Figure 4 WT 2 pg/pL Tm (2 hr) I Disi -+1-+ WT7 Tis Disi IDis + + IVI DisVlDis VIII + -+ IXi D isX bisXI\iDisXViDisXVI 2 pg/pL Tm (2 hr) 62 Although aneuploid yeast strains can mount a heat-shock response, the proteotoxic stress that we observe in disomic cells under standard growth conditions (25*C, YEPD) is not sufficient to induce a canonical heat-shock response in most disomes (Figure 5) (Torres et al. 2007). This lack of a heat shock response is expected, as this response is tailored to an acute stressor and is transient in nature (Gasch et al. 2000b). In contrast, aneuploidy is a chronic stress and adaptation to the aneuploid state may have taken place. It is however noteworthy that disomes IV and XIV, which adapt to heat-shock with the same kinetics as wild-type cells (Figure 1F, 2) upregulate genes involved in the heat-shock response even under normal growth conditions (Figure 5). Analysis of the abundance of the chaperones Hsp104, Ssal, Sse2 and Hsp42 further confirmed the absence of a canonical heat-shock response (Figure 6). Although aneuploidy is not sufficient to induce the canonical heat-shock response, most disomic yeast strains show a transcriptional response reminiscent of the environmental stress response (Torres et al. 2007), which encompasses a subset of the heatshock response Gasch et al. 2000a). HSP1O4 RNA levels, for example, increase with degree of aneuploidy (Sheltzer et al. 2012). Taken together, out results indicate that disomic yeast strains experience proteotoxic stress that is evidenced by increased aggregate burden, both under normal growth conditions and under conditions that induce proteotoxicity. 63 Figure 5: Heat shock signature in aneuploid strains grown in normal, unstressed conditions. Microarray data from aneuploid yeast strains grown in -His G418 medium at 25*C from Torres et.al. (Torres et al. 2007) were mined for genes shown to be upregulated (A) and downregulated (B) in the heat-shock response (Gasch et al. 2000b). A Heat Shock Score was calculated by averaging the log2 disome/wt ratio of all heat shock induced or heat shock down-regulated genes. Shown are the averages of all replicates available and the SEM. Note, at present it is unclear why disomes XIII and XIV exhibit increased expression of genes upregulated in response to heat-shock but not of genes down-regulated upon heat-shock. 64 Figure 5 A 1.0- K 0.80.6- Ci) 0.40 c) g) - CL0 0.20.0' FIW.I nn -,-uzL 1-I H rL rL 1, + co "C B 0.20.0CD-0.2- iuu Yy -40.4- ri rT-, Z0, (2-0.6< 0'-0.81 .0 IT \A 40,\-x 65 Figure 6: The abundance of several chaperones is unaltered in many disomic yeast strains. Hsp104, Ssal, Sse2 and Hsp42 protein levels were examined in wild-type cells and disomic yeast grown at 25*C in YEPD. Pgkl was used as a loading control. (a-g) Hspl04-eGFP levels were determined in WT (A31392), Dis I (A31393), Dis II (A31394), Dis IV (A31395), Dis V (A31396), Dis VIII (A31397), Dis IX (A31398), Dis X (A31399), Dis XI (A31400), Dis XII (A31401), Dis XIII (A31402), Dis XIV (A31403), Dis XV (A31404), and Dis XVI (A31405) in 2-fold dilutions. (h) Ssal-3HA levels were determined in WT (A32407), Dis II (A32408), Dis V (A32409), Dis VIII (A32410), Dis X (A32411), Dis XI (A32412) Dis XIII (A32413), Dis XV (A32414) and Dis XVI (A32415). (i) Sse2-3HA levels were determined in WT (A32416), Dis V (A32417), Dis VIII (A32418), Dis X (A32419), Dis XIII (A32420), Dis XV (A32421) and Dis XVI (A32422). (j) Hsp42-3HA levels were determined in WT (A32423), Dis II (A32424), Dis VIII (A32425), Dis XIII (A32426), Dis XIV (A32427) and Dis XVI (A32428). 66 Figure 6 A WT x Dis I Dis 11 x x xxx x xx x H - > 5; OL5 Hspl04-eGFP -- . - Pgkl B WT X)( Dis IV x aoqrNj- x x ooqwNj- x I,- Hspl04-eGFP Pgk1 C Pgk1 I Dis V XX xxxx 00oV(N- .. Ssa1-3HA 0 -- 7 -- I Sse2-3HA --- -- --- 1 Pgkl -- -- *mom 1 Disv III WT Hspl04-eGFP Pgk1 WI D ~xx xx cqwCN Dis X F- ;A - I 0 V Hsp42-3HA Pgkl Dis XI x x xx x 4x x 0 r- - 00 rq -- - 144000 I HsplO4-eGFP II Pgkl E WT x Dis XII x x x x Dis XIII xx x x xx IIHsp104-eGFP " "IWi S F WT 00 x 4x CN x '- iss Dis XIV Dis XV x x xx 00 V N Pgkl x xx x O - V~ CN 1 Hsp104-eGFP i 40wm--- - -*wow* % Pgk1 MO"" G ~x WT Dis XVI x4x xx mi x EIb qmpow*wow I I Hsp104-eGFP I Pgkl 67 Meiotic and mitotic chromosome mis-segregationleads to protein aggregateformation. Increased protein aggregation was not only observed in strains harboring single chromosomal aneuploidies, but also in aneuploid cells that arose from meiotic and mitotic non-disjunction. Triploid cells induced to undergo meiosis produce highly aneuploid progeny with karyotypes ranging from diploid to highly aneuploid (St Charles et al. 2010). The majority of the aneuploid progeny is inviable (Parry and Cox 1970), but some genetically unstable aneuploid strains can be obtained (Sheltzer et al. 2011; Zhu et al. 2012). As colony formation is a prerequisite for the analysis, we were only able to analyze those aneuploids that were healthy enough to form colonies. Nevertheless, analysis of 19 products of triploid meioses showed that the percentage of cells harboring Hspl04-eGFP foci was increased in most strains (Figure 7A). Chromosome mis-segregation during mitosis also resulted in aggregate formation. Strains harboring temperature-sensitive alleles of genes encoding the kinetochore component Ndc1O or the Aurora B kinase Ipli were arrested in G1 and released to progress through the cell cycle at the semi-permissive temperature of 30'C. Under these conditions, 35% of ndclO-1 and 29% of ip/1-321 cells were unable to segregate a GFP-marked chromosome IV (Figure 7B), indicating that dramatic chromosome mis-segregation occurs under these growth conditions. Aggregate formation was increased as early as 3 hours after release from the pheromone-induced G1 arrest (Figure 7C). Importantly, this increase in Hspl04-eGFP foci depended on cell division. When ndclO-1 or ipll-321 cells were induced to undergo a synchronous cell cycle at 30'C but chromosome segregation was prevented by treating cells with the microtubule-depolymerizing drug nocodazole, Hspl04-eGFP focus number did not increase (Figure 3C). We conclude that the percentage of cells harboring 68 Hspl04-eGFP-decorated protein aggregates is increased in most, if not all, aneuploid strains and that aggregates form soon after chromosome non-disjunction. Figure 7: Meiotic and mitotic chromosome non-disjunction causes increased Hspl04-eGFP focus formation. (A) The percentage of cells with Hspl04-eGFP aggregates was analyzed in progeny of diploid (A28220) or triploid (A28219) strains 3 days after germination (n=100 cells/strain). Note that although many of the progeny from the triploid meioses will harbor multiple aneuploidies, some will also be euploid or will have become euploid as they proliferate. (B, C) Wild-type (A5244), ndclO-1 (A28204) and pll-321 (A16154) mutants harboring a GFP-marked chromosome IV, and wild-type (A25654), ndclO-1 (A27681) and ipll-321 (A27682) mutants harboring Hspl04-eGFP, were arrested in GI with pheromone at 25*C followed by release at 30*C either in the presence (10pg/ml; Arrested) or absence (Dividing) of nocodazole. Samples were taken after 3 hours to determine the percentage of cells that correctly segregated chromosome IV (B) and that harbored Hspl04-eGFP foci (C). We note that the temperature shift during this experiment may inflate the percentage of cells harboring aggregates in all strains, as they will be adapting to the temperature shift. 69 Figure 7 A .5 -c 4.J 40 4- 908070- 0 60- C-) C 5040- C', 0 1- C- B A 3020100 AA A JA AA A U .mE U *. 'A AAA A A 6".. A I Diploid F1 Triploid F1 100 L) 75. cu 50. 0 0 CD> 25. <n 0 WT C 60- .U 4- -c 0L U- ndc1O-1 ipi 1-321 Dividi ng Arres ted ** 40- C', C-) 0. 0 C) 20- 0- WT ndc1O-1 ipl1-321 70 A neuploid strainsfail to efficienty fold the protein quality controlsensor VHL Protein aggregates in disomic yeast strains could be the result of proteins generated from the additional chromosomes overwhelming and/or impairing chaperones. To test this idea we challenged the cell's protein quality-control pathways using the well-studied substrate, the human von Hippel-Lindau protein (VHL). VHL is unable to fold without its binding partners ElonginB and ElonginC. When human VHL is expressed in yeast in the absence of Elongin B and C, the protein is quickly ubiquitinated and degraded (McClellan et al. 2005; Kaganovich et al. 2008) (Figure 8). The quality-control pathways involved in the elimination of misfolded VHL are known: folding-defective VHL is shuttled from Hsp70 to an Hsp90 complex that enables degradation by the ubiquitin-proteasome system (McClellan et al. 2005). When any of these pathways are defective, misfolded VHL forms aggregates that are seen as foci in cells expressing VHL as a GFP fusion (Kaganovich et al. 2008) (Figure 9F.) Disomic yeast strains grown under non-stress conditions (YEPRG, 25*C; Figure 9A) and under conditions of heat stress (2 hours at 37'C; Figure 9B) showed increased VHL focus formation; slow-growing mutants or strains carrying human DNA did not (Figure 9C, D). The failure to process misfolded VHL-GFP was not specific to the disomic strains, but was also observed in progeny of triploid meioses (Figure 9E). We note that haploid strains obtained from diploid meioses harbored a higher percentage of cells with VHL-GFP foci than the haploid control strain analyzed in the experiment shown in Figure 9A. We suspect that germination and colony growth on selective medium places an increased burden on the cell's protein qualitycontrol systems compared to growth in liquid rich medium (YEPRG) at 25*C. We conclude that targeting of VHL-GFP for proteasomal degradation is compromised in aneuploid cells, either because the protein quality-control pathways (chaperones and/or the proteasome) of 71 the cells are defective or they are functional but overwhelmed by changes in the cell's proteome caused by the aneuploid state. Figure 8: Inhibiting the proteasome causes VHL-GFP aggregate accumulation in aneuploid and euploid strains. Wild-type and disomic yeast strains deleted for the multidrug transporter PDR5 and expressing a VHL-GFP fusion were analyzed after a transient (2hr) exposure to the proteasome inhibitor MG132 (80M) and the percentage of cells with VHL-GFP foci was determined (n=3; SEM, n=100/time point). (a) Strain order: A32076, A32078, A32080, A32081, A32082, A32083, A32084, A32086, A32088, A32089 (b) Wild-type (A32076), cdc23-1 (A30461), cdc28-4 (A30462) and wild-type strains harboring YACs carrying human DNA (A29969 and 29971). Figure 8 A nnr 1000 >; LZ 75- 501 0 25- 0 B 10075 -C% 50 C CO - - - , , , , , , , , EM RM E I I 25 0 . 72 Figure 9: Aneuploid yeast display hallmarks of impaired protein quality control. (A) Wild-type and disomic yeast strains deleted for the multidrug transporter PDR5 and expressing a VHL-GFP fusion were grown in YEP 2%Raf 2%Gal at 25*C and the percentage of cells with VHL-GFP foci was determined (n=3; SEM, n=300 cells/time point; *P<0.05, **P<0.005; ***P<0.0005; Student's t test). Strain order: A32076, A32077, A32078, A32079, A32080, A32081, A32082, A32083, A32084, A32085, A32086, A32087, A32088, A32089 (B) Strains described in (A) were analyzed after a 2hr incubation at 37*C (n=100). (C, D) cdc23-1 (A30461), cdc284 (A30462) and wild-type strains harboring YACs carrying human DNA (A29969 and A29971) were grown at 25'C (C) or shifted for 2 hours to 37*C (D) to analyze VHL-GFP focus formation. (E) The percentage of cells with VHL-GFP foci was determined in progeny of diploid (A28388) or triploid (A28389) strains 4 days after germination (n=100/colony). (F) Images of VHL-GFP aggregates. Aggregates are in green, DNA in blue. 73 Figure 9 A C 17.515.0- aL' ) 10.0C-4 15.0 ,,** ,*** I 12.5- I 17.5 .g 5 * 7.5- 5.0o 2.5- LL 9 12.5- 1 10.0 6 7.5 ._' 5.0 S 2.5-- 0 0 D t21 C 0.0' B a 807060- >0 I , 5040- 404- C') E 9 * . 0 * >U) 3020100 0 - F 9080- A A 70 A 60 U GFP GFP & DAPI a- AA AA AA * -J 3020100 0 70 60 50 40 30 -~ 20 10 0 Q-ii * * A 0UDiploid F1 Triploid F1 74 Loss of UBP6 reduces aggregate burden in disomicyeast strains. The high incidence of both endogenous and VHL protein aggregates in aneuploid strains suggests that aneuploidy negatively impacts protein folding and/or degradation of misfolded proteins. Previous studies showed that ubiquitin-proteasomal degradation is important for the survival of aneuploid yeast strains (Torres et al. 2007; Torres et al. 2010). Insufficient proteasome activity could also be responsible for increased aggregate formation in aneuploid cells. A prediction of this hypothesis is that increasing proteasome function decreases aggregate burden in aneuploid cells. To test this possibility we examined the consequences of deleting UBP6 on Hspl04-eGFP focus formation in disomic yeast strains. Ubp6 associates with the proteasome and removes ubiquitin chains from substrates. This not only allows for the recycling of ubiquitin but also causes proteasome substrates to escape degradation. This is evident from the analysis of cells lacking UBP6. Degradation of all proteasome substrates analyzed to date is accelerated in such cells (Hanna et al. 2006; Peth et al. 2009). We deleted UBP6 in disome V and disome XI cells, whose proliferation improves when UBP6 is deleted, and in disome II cells, in which deleting UBP6 leads to decreased proliferation (Torres et al. 2010). Deletion of UBP6 reduced aggregate burden in all three disomic strains (Figure 10). This finding is consistent with our previous observation that deletion of UBP6 causes attenuation of levels of proteins with high relative expression in all disomic strains analyzed, irrespective of whether deletion of UBP6 improves proliferation (Torres et al. 2010). Interestingly, in disome V strains, aggregate burden was reduced to almost wild-type levels when UBP6 was deleted (Figure 10). This finding raises the interesting possibility that the increased proliferative abilities of disome V ubp6D cells are due to a reduction in protein aggregates. We conclude that enhanced proteasomal degradation reduces the aggregate burden in all disomic strains analyzed. 75 Figure 10: Increased proteasome activity decreases aggregate burden in disomic strains. Wild-type(A3369), disome II (A33370), disome V (A33371) and disome IX (A33372) cells harboring a deletion of UBP6 and the HSP104-eGFP fusion were grown to exponential phase in YEPD, and the percentage of cells harboring Hspl04-eGFP foci was determined n=3; SEM, n=100 cells/time point. Figure 10 .5 402 6 D UBP6 0 I]ubp6 Cj) 0 WT Dis 11 Dis V Dis XI Hsp90 folding capacity is reduced in many disomicyeast strains. Are other protein quality-control systems also affected in aneuploid cells? Because VHL is an Hsp90 client, we explored the in vivo folding activity of Hsp90 and found it to be reduced in many disomic strains. Hsp90 is a highly abundant chaperone that, in concert with co-chaperones, folds cytosolic proteins (McClellan et al. 2007; Franzosa et al. 2011). Consistent with previous results using the Hsp90 inhibitor geldanamycin (Torres et al. 2007), we found that several disomic yeast strains are more sensitive to the Hsp90 inhibitor radicicol than the euploid control strain (Figure 11A). 76 To examine Hsp90 activity, we analyzed the in vivo folding activity of the well-studied Hsp90 model substrate, the tyrosine kinase Src. Both c-src and the oncogenic form, v-src, depend on Hsp90 for folding (Kimura et al. 1995; Nathan et al. 1997). For unknown reasons, overexpression of v-src, but not c-src, is lethal in budding yeast (Xu and Lindquist 1993). Compromising Hsp90 activity suppresses v-src folding and activity and, consequently, this lethality (Nathan and Lindquist 1995; Nathan et al. 1997) (Figure 11B). We found that the toxicity of v-src was diminished in many aneuploid strains (Figure 11C), suggesting a reduction in Hsp90 activity. To further explore the activity of v-src, we took advantage of the low levels of endogenous tyrosine phosphorylation in yeast that are dramatically increased when v-src is expressed from the galactose-inducible GALI -10 promoter (Figure 11D). Total tyrosine phosphorylation was reduced in disomes II, V, VIII and XII (Figure 11D), correlating well with these cells' ability to form colonies under v-src-inducing conditions (Figure 11C). The inability to generate active v-src was not due to decreased mRNA expression, as v-src RNA levels were as high or higher in the disomes than in wild-type cells (Figure 11 E). Reduced v-src activity was also observed in disomes I, XIII, XV and XVI as judged by reduced tyrosine phosphorylation levels, but this decreased activity was not sufficient to allow growth on v-src-inducing medium (Figure 11 C, D). Our results show that 8 out of 11 disomes exhibit reduced Hsp90 activity. Hsp90 may be overloaded by substrates that rely on this chaperone to be folded. It is also possible that the activity of the Hsp90 folding machinery is reduced. Given that many different disomic strains exhibit decreased Hsp90 activity, we favor the idea that the Hsp90 folding reservoir is depleted, rather than inactive, in aneuploid strains. Hsp90 is thought to serve a limited number of clients under normal growth conditions and to be present in excess (Borkovich et al. 1989; Neckers 2007). It is therefore surprising that Hsp90 activity appears limiting in many disomic strains. 77 Perhaps under conditions of proteotoxic stress, Hsp90's folding repertoire is expanded. We conclude that many aneuploid cells experience saturation of the Hsp90 system. Figure 11: Hsp90 folding capacity is limiting in many disomic strains. (A) Disomic yeast harboring a deletion of the multidrug transporter PDR5 were grown in YEPD or YEPD containing 70 ptM radicicol at 30'C to determine their doubling time. Mean and SEM of 3 replicates is shown. Strains in order are: A15549, A15551, A15553, A15555, A15557, A15559, A15561, A15563, A15566, A15567, A15569, A15571, A15573. (B) Schematic of v-src/c-src Hsp90 assay. Hsp90 is required to fold c-src and toxic v-sr. A reduction in Hsp90 activity results in misfolded v-src and restores cell viability. (C) Wild-type and disomic yeast strains carrying c-src or v-src under the galactose-inducible CAL1-10 promoter were grown under conditions where expression is repressed (-URA 2% Glu.) or induced (-URA 2% Raf. Gal.). 10-fold dilutions were plated. C-src strains in order are: A32090, A32091, A32092, A32093, A32094, A32095, A32096, A32097, A32098, A32099, A32100, A32101. V-src strains in order are: A32102, A32103, A32104, A32105, A32106, A32107, A32108, A32109, A32110, A32111, A32112, A32113. (D, E) Wild-type and disomic yeast strains harboring the GAL-v-src fusion, were grown in YEP+ 2% raffinose. Galactose was added and the relative amount of v-src RNA (E) and total tyrosine phosphorylation (D) was determined before and after 2 hours of v-src induction. Vsrc strains in the same order as in (C). 78 Figure 11 A B 25- .C20- EJ YPD csrc 0 70 pM Radicicol csrc +,0 i! E 15 10- -ut- -. -I. W4 D WT Dis Dis Dis Dis Dis 2 2 0 2 II 0 V 2 0 2 VIII IX 0 2 0 2 WT Dis Dis Dis Dis Dis Dis XIV XV VI XI XII 0 210 210 210 2 0 202 0 2 m. *.1wM Z . <4- 2.5 0 hr induction 2 hr induclion 1.5- S2- 1.0. 0.5 0 41 0 hr induction 2 hr induction 2.0 E 3- \+4 -+- Misfold csrc Viable Viable vsrc + Hsp9O -+ Fold vsrc vsrc +060 Misfold vsrc Viable - II III 0.11 . 1 .9. Ell lz qAC., 4z : i WT csrc WT vsrc Dis I csrc Dis I vsrc Dis 11 csrc Dis 11 vsrc Dis V csrc Dis V vsr WT csrc WT vsrc Dis X11 csrc Dis XII vsrc Dis XIII csrc Dis XIII vsrc Dis XIV csrc Dis XIV vsrc E 5-E Fold csrc WT csrc WT7 vsrc Dis Vill csrc Dis Vill vsrc Dis IX csrc Dis IX vsrc Dis X csrc Dis XI vsrc PpY Kar2 11 -+ C 0 0 hr post induction 0 + Hsp9O P, 0 0 WT csrc WT vsrc Dis XV csrc Dis XV vsrc Dis XVI csrc Dis XVI vsrc 79 A neuploid strainsare more susceptible to protein aggregatesassociatedwith human disease. Does aneuploidy also cause cells to be more susceptible to protein folding defects associated with human disease? To address this question, we employed an assay that measures the activity of the prion protein Sup35 and assessed toxicity associated with the glutamine-rich protein Httl in disomic yeast strains. The prion [PSI+] is formed by Sup35, a subunit of the translation terminator complex. When Sup35 switches to the aggregated amyloid conformation [PSI+], much of the protein becomes unavailable to terminate translation, causing read-through of stop codons (Liebman and Sherman 1979). Because the basal conversion frequency of Sup35 to its prion form is low (10-1-107), we used strains carrying a variant of the SUP35 gene (SUP35-R2E2) that increases the conversion frequency (Liu and Lindquist 1999; Cox et al. 2003) to study the effects of aneuploidy on SUP35 activity. We then utilized an assay where conversion from [psi-] to [PSI+] results in read-through of three stop codons upstream of GFP, allowing expression of the fluorescent protein (Tyedmers et al. 2008). Single colonies obtained from a frozen stock were inoculated into rich medium, and the percentage of fluorescent cells was determined immediately after inoculation and after 8 and 24 hours. All disomic strains tested showed an increase in the fraction of cells expressing GFP (Figure 12A, B, Figure 13). Attempts to visualize the Sup35 aggregates by SDD-AGE (SemiDenaturing Detergent-Agarose Gel Electrophoresis) were unsuccessful. We therefore cannot exclude the possibility that mechanisms other than prion conversion lead to the observed increase in the percentage of GFP-positive cells in the disomic strains. We, however, favor the interpretation that Sup35 aggregates are in the detergent-soluble, small oligomer stage that precedes the large amyloid aggregates detectable by SDD-AGE (Halfmann et al. 2010) 80 and/or that aggregates comprise a small fraction of total Sup35 protein and are thus undetectable by this technique. Figure 12: Disomic yeast strains shown increased expression of the Sup35 prion reporter. Single colonies of wild-type and disomic strains carrying the SUP35-R2E2 allele and a GFP construct preceded by 3 stop codons were inoculated into SC medium and the percentage of fluorescent cells was determined after 0, 8 and 24 hours by flow cytometry (A). Shown is the ratio of GFP+ cells after 8 and 24 hours of growth to GFP+ cells immediately after inoculation (0 hr). The mean and SEM of at least 12 single colonies are depicted. Strains in order are: A31114, A29843, A29845, A29846, A29847, A29848, A31110, A31111, A31112, A29450, A31113. (B) Strains in (A) and A22361 (no GFP control) were grown for 24 hours and total protein was extracted. GFP protein levels were analyzed by Western blot analysis. Pgkl was used as a loading control. 81 Figure 12 A 12U) C/) 0~ 4. 0 E8hr/Ohr M24hr/Ohr 11 I Isin 4 It liftni ill B GFP I Pgkl 82 Figure 13: Sup35 activity in disomic yeast strains. Single colonies of wild-type and disomic strains carrying the SUP35-R2E2 allele and a GFP construct harboring 3 stop codons were inoculated into YEPD medium and the percentage of fluorescent cells was determined immediately after inoculation and after 8 and 24 hours of growth by FACS. The average of at least 12 single colonies is shown and data are plotted as a percentage of GFP positive cells of a total of 30,000 cells/sample. Strains in order are: A31114, A29843, A29845, A29846, A29847, A29848, A31110, A31111, A31112, A29450, A31113. Figure 13 100- 80- U) 04 L1 Time =0 Time = 8 Time = 24 60- 4 20- 0goEl 83 Huntington's disease is a neurodegenerative disease associated with P-sheet aggregates comprised mainly of the Huntingtin (Httl) protein (Goedert et al. 2010). Toxicity and disease phenotypes require that the poly-glutamine (polyQ) stretch in its N terminus expand beyond 38 repeats (Duyao et al. 1993; 1993). We used yeast strains expressing a 17 amino acid fragment of Httl exon 1 with polyQ tracts of varying length to determine the susceptibility of aneuploid yeast to expanded polyQ stretches (Duennwald et al. 2006). In euploid cells, Httl harboring 25 glutamine residues (25Q) is not toxic when expressed from the galactose-inducible CALi-10 promoter, but Httl harboring 46 or 72 Qs causes toxicity (Duennwald et al. 2006). All disomes tested, except for disome VIII, exhibited increased sensitivity to Htt1-polyQ expression compared to the euploid control (Figure 14A). Httl46Q-CFP aggregates are also accumulated more readily in many of the disomic strains analyzed (Figure 14B). The lack of sensitivity of disome VIII is most likely due to reduced expression of the construct (Figure 14C). We conclude that the proteotoxicity that afflicts aneuploid yeast cells can predispose them to the accumulation of protein aggregates associated with human diseases. 84 Figure 14: Disomic yeast strains exhibit increased sensitivity to Huntingtin polyQaggregates. (A) Wild-type and disomic strains harboring a CAL-FLAG-HTT1(17AA)25QApro-CFP, GAL-FLA C-HTF T(17AA)46QApro-CFP or GAL-FLA G-HTT(17AA)72QApro-CFP construct were grown under conditions where expression is repressed (YEPD) or induced (YPRG). 10-fold dilutions were plated. 25Q strains in order are: A32114, A32115, A32116, A32117, A32118, A32119, A32120. 46Q strains in order are: A32121, A32122, A32123, A32124, A32125, A32126, A32127, A32128. 72Q strains in order are: A32129, A32130, A32131, A32132, A32133, A32134, A32135. (B) Wild-type and disomic strains harboring the GAL-FLA G-HTT(17AA)46QApro-CFP construct were grown for 8 hours in the presence of galactose to determine the percentage of cells with Httl-46Q-CFP foci (n=100). Shown are mean and SEM of 3 independent experiments. (C) Expression of the CAL-HTIT1(17aa)-FLAG- n9-CFPconstructs. Strains were grown in YEP 2% raffinose to OD 600= 0.2 when 2% galactose was added. RNA was extracted from samples taken after two hours and the amount of H7T1-nQ-CFP RNA was determined via Northern blot analysis. 85 Figure 14 A OFF ON OFF ON OFF ON WT Dis 11 Dis V Dis Vill Dis XI Dis XIll Dis XVI 25Q B 72Q 30- U0 0 46Q I 20- 6 ~10- 0~ 0 ~%>~' C ' A I1 VV 1 Dis Dis Dis Dis Dis Dis 11 1 V IilI Al IAIi JAVI hours 0 21 0210 210 210 210 202 -N Vqs post nd. 25Q rRNA 1 0 *0 46Q NS rRNA 4 I 0* 9 p 72Q cam en~ amerRNA 86 Discussion Our studies of aneuploid yeast have revealed the dramatic effect of an unbalanced karyotype on cellular protein homeostasis. All aneuploid strains, irrespective of how they were generated or their karyotype, showed an increased protein aggregate burden. Aneuploid strains are prone to aggregation of endogenous proteins as well as of ectopically expressed hard-to-fold proteins such as polyQ stretch-containing proteins. We do not know which proteins comprise the aggregates observed in aneuploid cells. Obligate chaperone clients present in excess due to aneuploidy could accumulate and then either form aggregates themselves or interfere with the folding of other chaperone clients. Identifying aggregate constituents will distinguish between these two non-mutually exclusive possibilities. Two protein quality-control systems, the proteasome and the Hsp90 chaperone, appear to be limiting in many aneuploid yeast strains. Increasing proteasome function by deleting UBP6 led to a decrease in aggregate burden in all aneuploid yeast strains analyzed. We also found that the Hsp90 substrate v-src was less active in many disomic strains indicating that Hsp90 activity is limiting, which could contribute to aggregate formation in these strains. This latter result is surprising as Hsp90 is highly abundant and its activity thought to be in excess in cells (Borkovich et al. 1989; Neckers 2007). Perhaps this is not the case, especially under conditions of proteotoxic stress. Hsp90 may not be the only chaperone system limiting in aneuploid cells. We speculate that other protein folding pathways are also saturated with different aneuploidies impacting different chaperone families to varying degrees depending on the identity of the proteins encoded on the extra chromosomes. Examining the activity of the different folding pathways in different disomic yeast strains will test this idea. 87 Why are aneuploid cells aggregate-prone? Aggregates could be a result of overwhelmed folding pathways, or they could stem from reduced chaperone activity. Both alternatives are possible, but given that the aneuploid chromosomes are actively expressed, it is likely that excess proteins produced from the aneuploid chromosomes occupy chaperones and thereby reduce their availability to assist in the folding of their other clients. What determines the extent of aggregate formation is not yet known. It does not appear to correlate with either the degree of aneuploidy (by DNA content), total protein in excess, delay in G1 or doubling time. However, it is important to bear in mind that our aggregate analyses do not measure absolute amounts of aggregated proteins in cells nor are they able to distinguish toxic from non-toxic aggregates. The environmental stress response, which encompasses part of the heat-shock response, correlates with degree of aneuploidy (Torres et al. 2007) (Sheltzer et al. 2012). We propose that protein aggregate burden correlates with the number of obligate chaperone clients, and hence with the distribution of their encoding genes in the genome. It may seem surprising that the cell's protein quality-control pathways cannot compensate for the presence of a single additional chromosome, which depending on chromosome size, results in 2 - 12 percent of the genome being imbalanced. Previous studies showed that even small amounts of misfolded proteins place a burden on the cell's protein quality-control systems and hence adversely affect cellular fitness. Expression of a single misfolded cytosolic protein at less than 0.1% of total protein leads to a significant decrease in proliferative abilities and the induction of a cytoplasmic unfolded protein response (Geiler-Samerotte et al. 2011). Importantly, the generation of misfolded proteins requiring the assistance of the cell's protein quality-control pathways is a common occurrence in aneuploid cells. It is well 88 established that many subunits of protein complexes only acquire a stable conformation by binding to other subunits of the complex (Imai et al. 2003; Boulon et al. 2010). Thus, every single polypeptide produced by genes located on aneuploid chromosomes that normally has a binding partner is - in the disomes - in excess. For example, if in euploid cells 1 percent of a subunit of a heterodimeric protein complex is present in excess due to variability in subunit expression and must be eliminated, the number of proteins that needs to be eliminated rises to 102% in cells that carry an additional copy of the gene encoding one of the two subunits. This scenario applies to all proteins encoded on the extra chromosome that require a binding partner to acquire a stable conformation. This dramatic change in protein stoichiometries, we propose, leads to an increased burden on the protein quality-control pathways of the cell. Individual subunits present in excess require the continuous assistance of chaperones, preventing chaperones from assisting other folding reactions and reducing the general folding capacity of the cell and thus interfere with their essential function of mediating folding of essential proteins (Hard et al. 2011). The fact that the aggregate phenotype was ameliorated when the ratio of uncomplexed proteins to properly complexed proteins was decreased by increasing base ploidy (as in trisomic strains) suggests that the proteotoxicity observed in aneuploids is indeed in part the result of the protein stoichiometry imbalances caused by aneuploidy, although it is also possible that diploid cells are more efficient at clearing aggregates. We furthermore propose that an additional burden on the protein quality-control machinery is generated by the overproduction of proteins encoded on the extra chromosomes that require chaperones for their function, such as protein kinases and WD40 repeat proteins. In summary, aneuploidy impacts protein homeostasis in multiple ways, so that even small unbalanced chromosomes have a significant impact on the cell's protein quality control systems. 89 A neuploidy in cancerand neurodegenerative diseases. Our results have important implications for how we think about the impact of aneuploidy on human disease. Solid tumors, which are highly aneuploid, have long been deemed chaperone-addicted (Neckers 2007; Workman et al. 2007; Powers et al. 2008). Eliminating HSF1, the master regulator of the heat-shock response, results in a lower incidence of tumors in mice (Dai et al. 2007). This dependence on chaperones has been attributed to the importance of chaperones for the folding of oncogenes. Our studies suggest that the aneuploid nature of tumors contributes to their dependence on chaperones such as Hsp90 for survival. We further suggest that aneuploidy could contribute to neurodegenerative diseases such as Huntington's or Alzheimer's Diseases. The human brain is a naturally aneuploid organ, with one third of fetal neurons and 10% of adult neurons being aneuploid (Rehen et al. 2001; Rehen et al. 2005; Yurov et al. 2007a; Yurov et al. 2007b). Our finding that aneuploidy causes proteotoxic stress including polyQ aggregate formation, raises the interesting possibility that aneuploidy reduces the cell's capacity to eliminate protein aggregates and/or increases the propensity for aggregate formation. Thus, their aneuploid nature may predispose neurons to protein aggregation diseases. Interestingly, mice chimeric for trisomy 16 (one of the mouse models of Down's syndrome) have been previously associated with increased susceptibility and poor prognosis when injected with the Scrapie prion protein (Epstein et al. 1991). Further studies of the proteotoxicity associated with aneuploidy could therefore provide important insights into tumorigenesis and neurodegenerative diseases and may even pave the way for the development of novel treatments for these diseases. 90 Materials and Methods Strains andplasmids: Strains used in this study are described in Table S1 and are derivatives of W303. Strains were constructed using PCR-based methods described by Longtine et al. (Longtine et al. 1998). The generation of disomic strains has been described previously (Torres et al. 2007). Karyotypes of all disomic and trisomic strains were confirmed by comparative genome hybridization (Torres et al. 2007). YACs used in this study have been previously described (Foote et al. 1989). The pGAL-VHL-GFP fusion is described in Kaganovich et. a. (Kaganovich et al. 2008). CA L-HYT1(I7aa)-FLAG-25Q-CFP and CA L-HT1(I7aa)-FLA G46Q-CFP and GAL-HT1(17aa)-FLAG-72,Q-CFP are described in Duennwald et aL (Duennwald et al. 2006). A naysis of endogenousprotein aggregatesin disomicyeaststrains. For analysis of endogenous aggregates, strains carrying an Hspl04-eGFP fusion were grown in YEPD medium. Exponentially-growing cells were fixed in 3.7% formaldehyde by adding 0.1 ml 37% formaldehyde to 1 mL of cells. Cells were then permeabilized in 1% Triton/Potassium Phosphate, washed and resuspended in KPi/Sorbitol. The percentage of cells harboring Hspl04-eGFP foci was determined in at least 100 cells per sample. Foci were defined as GFP dots that were visible without the aid of a camera. A na/ysis of endogenous aggregatesin progenj of diploid and trpiloidmeioses. Diploid and triploid cells were sporulated. Tetrads were dissected on YEPD plates. Colonies that grew up were diluted in water and Hspl04-eGFP foci were analyzed as described above 91 in at least 100 cells/colony. We note that since this analysis relies on colony growth, the most severe aneuploids that cannot form colonies cannot be analyzed. A na/ysis of endogenous aggregates upon chromosome mis-segregation. Haploid strains harboring the temperature-sensitive ndclO-1 or alleles were arrested /pl1-321 in YEPD + lOg/ml a-factor at room temperature. After 90 minutes, 5pg/ml a-factor was added. 180 minutes after the initial a-factor addition, cells were washed and released into pre warmed (30*C) YEPD. To half of the culture 15 pig/ml nocodazole was added. Hspl04eGFP foci were analyzed 3 hours after release from the G1 arrest as described above. High temperature adaptationtime courses. Cells were grown to exponential phase in YEPD at 25*C. Cells were collected by filtration and resuspended in pre-warmed (37*C) YEPD. Samples were taken at the indicated times (Oh is immediately before shifting temperature). Hspl04-eGFP foci were counted in at least 100 cells/time point as described above. A naysis of cellularproteinqualiy control using the VHL-GFP reporter. Strains deleted for the multidrug transporter PDR5 harboring the GZAL-VHL-GFP fusion and split in two. Half were grown at 25*C in YEP 2% Raffinose 2% Galactose to OD e0=0.2 6 the culture was maintained at 25*C and the other half was shifted to 37'C. Samples were taken 2 hours later, fixed with 3.7% formaldehyde and the percentage of cells harboring VHL-GFP foci was determined. GFP foci were counted without the aid of a camera, and any cell with a visible focus was counted as a cell harboring a focus. At least 300 cells were counted for cultures grown at 25*C and at least 100 for cultures grown at 37*C. 92 A naysis of VHL aggregates in progeny of diploid and triploidmeioses. Diploid and triploid cells were sporulated and tetrads were dissected on plates lacking leucine containing 2% raffinose and 2% galactose. Colonies were resuspended in water and the percentage of cells with VHL-GFP foci was determined as described above. As this analysis relies on colony growth, the most severe aneuploids that cannot form colonies cannot be analyzed. Effects of radicicolon the growth rate ofdisomicyeast strains. Disomes deleted for the multidrug transporter PDRS were inoculated at OD60 0 =0.1 in YEPD in 96-well plates either lacking or containing 70ptM radicicol in freshly made medium. OD 60 was measured every 15 minutes on a plate reader (Synergy2, Biotek) for 24 hours. Doubling times were calculated using the exponential growth phase of each culture. Assessing v-src and Httl-poyQ glutamine toxicity: Tenfold serial dilutions were prepared and spotted onto the appropriate medium: medium lacking uracil and containing either 2% glucose or 2% raffinose, 2% galactose for GAL--src, GAL-c-src containing strains and YEPD or YEP 2% Raf 2% Gal for GAL-HTT1(17aa)FLA C-nQ-CFPharboring strains. Plates were imaged after 3 days of growth at 25 0 C. Western blot analyses. Cells were harvested by adding an equal volume of 10% trichloroacetic acid to the cell culture and incubated on ice for at least 20 minutes. Cells were then washed with 1.5mL of acetone. The dried pellet was resuspended in 100 pLL of protein breakage buffer (50mM Tris, 93 pH 7.5, 1mM EDTA, 2.75 mM DTT, and Roche Complete protease inhibitor, used per the manufacturer's instructions). 100 ptL of glass beads were added and the cells broken by beating for 2.5 minutes on a Biospec mini-bead beater. 50 piL of 3X SDS sample buffer were added, the samples boiled for 5 minutes, then centrifuged for 5 minutes. An equal volume of lysate was loaded onto 10% SDS polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. Hspl04-eGFP was detected using a mouse anti-GFP antibody (L8, Clontech) at a 1:1,000 dilution. Pgk1 was detected using a mouse anti-Pgkl antibody (A6457, Molecular Probes) at a 1:5,000 dilution. Ssal-3HA, Sse2-3HA and Hsp42-3HA were detected using a mouse anti-HA antibody (HA.11, Covance) at a 1:1,000 dilution. Total phosphotyrosine levels were detected using a mouse anti phosphotyrosine antibody (4G10, Millipore) at a 1:1,000 dilution. The secondary antibody was a sheep anti-mouse antibody coupled to horseradish peroxidase (NA931, GE Healthcare) and used at a 1:2,000 dilution. Kar2 was detected using a rabbit anti-Kar2 antibody at a 1:200,000 dilution and followed by donkey anti-rabbit antibody coupled to horseradish peroxidase (NA9340, GE Healthcare) used at a 1:2,000 dilution. Bands were detected using Amersham ECL Plus Substrate according to the manufacturer's instructions. Determination of Sup35function. The GFP read-through assay to assess Sup35 function was performed essentially as described previously (Tyedmers et al. 2008). Briefly, strains were streaked from frozen stocks on selective medium (-His, G418) and allowed to grow to single colonies for 3 days. Single colonies were then resuspended in 200 p1 SC, and 100 p was analyzed immediately by flow cytometry to determine %GFP-positive cells (n>10,000). The remaining 100p were used to inoculate 3-ml SC cultures, which were grown at room temperature and maintained in 94 exponential phase. Samples were taken for flow cytometry analysis after 8 and 24 hours of growth and the percentage of GFP-positive cells was determined. A nafysis of lyQ aggregates. Strains harboring the GAL-HTT1(17aa)-FLAG46Q-CFPconstruct were grown at 25'C in YEP 2% Raffinose 2% Galactose to OD60 () =0.4. Samples were taken, fixed with 3.7% formaldehyde and the percentage of cells harboring polyQ-CFP foci was determined. CFP foci were counted without the aid of a camera, and any cell with a visible focus was counted as a cell harboring a focus. At least 100 were counted for each replicate. Cell Imaging. For the analysis of Hsp104-eGFP and VHL-GFP foci, cells were fixed in 3.7% formaldehyde by adding 0.1 ml of 37% formaldehyde to 1 mL cells. Cells were then permeabilized in 1% Triton/Potassium phosphate washed and re-suspended in DAPI /KPi/Sorbitol Microscopy was performed using a Zeiss Axioplan 2 microscope with a Hamamatsu OCRA-ER digital camera. Image analysis was performed with Openlab 4.0.2 software. Northern blot analysis. Total RNA was purified by phenol extraction and isopropanol precipitation as described in (Hochwagen et al. 2005). 10pg of total RNA were separated on a 1.1% agarose gel containing 6% formaldehyde and 40 mM MOPS (pH 7.0). Gels were blotted in 1OX SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH = 7.0]) onto Hybond-XL 95 membranes (Amersham Biosciences). Blots were probed overnight with radioactively labeled HA C1 or GAL-HTT1(17aa)-FLA C-nQ-CFPspecific probes. Heat Shock Microarrys. Strains were grown in YEPD at 25 0 C to OD 60 =0.2, collected by filtration and shifted to pre-warmed (37*C) YEPD. Samples were collected 0, 5, 15, and 30 min after temperature shift. WT grown at 25 0 C was used as a reference for all samples. RNA preparation and microarrays were performed as described previously (Torres et al. 2007). Briefly, total RNA was ethanol precipitated and further purified over RNeasy columns (Qiagen). 325ng of RNA were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Reactions were performed as directed except half the recommended reaction volume and one quarter the recommended Cy-CTP amount was used. Dye incorporation and yield were measured with a Nanodrop spectrophotometer. Equal amounts of differentially-labeled control and sample cDNA were combined such that each sample contained at least 2.5pmol dye. Samples were fragmented, combined with hybridization buffer, and boiled for 5 minutes, and applied to a microarray consisting of 60mer probes for each yeast open reading frame (Agilent). Microarrays were rotated at 60*C for 17 hours in a hybridization oven (Agilent). Arrays were then washed according to the Agilent SSPE wash protocol, and scanned on an Agilent scanner. The image was processed using the default settings with Agilent Feature Extraction software. All data analysis was performed using the resulting log 2 ratio data, and filtered for spots called as significantly over background in at least one channel. Data were normalized to account for the extra chromosomes as previously described (Torres et al. 2007). Data were mined for genes that comprise the heat-shock response Gasch et al. 2000a). 96 The full dataset has been deposited in the Gene Expression Omnibus under the accession: GSE40073. Acknowledgements We thank B. Vincent, D. Jarosz and M. Duennwald for reagents; J. Boulin for technical assistance; and S. Lindquist, F. Solomon, and members of the Amon Lab for suggestions and critical reading of this manuscript. This work was supported by the National Institute of Health (GM056800 to A.A) and a Ludwig Fund Graduate Fellowship (to A.O.). A.A is an investigator of the Howard Hughes Medical Institute. Strains used in this study. All straisn are of the W303 background Strain Number A22361 A6863 A6865 A24367 Disome A28265 V A27036 VIII A13975 IX A21986 X A28266 XI A12694 XII A21987 XIII - I II IV Relevant Genotype MATa, adel::HIS3, lys2::KanMX6 MATa, adel::HIS3, ade1::KanMX6 MATa, lys2::HIS3, lys2::KanMIX6 MATa, trpl::HIS3, trpl::KanMX6 MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHR007C::KanMX6 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6 MATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6 MATa, intergenic region (430900-431000) between YKLO06C-A and YKL006W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6 MATa, adel6::HIS3, ade16::KanMX6 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and 97 YMR018W::KanMX6 A28344 XIV A27930 A27096 A31392 A31393 A31394 A31395 XV XVI A31396 V A31397 VIII A31398 IX A31399 X - I II IV MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNL0O4W::KanMX6 MATa, leu9::HIS3, leu9::KanMX6 MATa, met12::HIS3, metl2::KanMX6 MATa, adel::HIS3, lys2::KanMX6, HSP1 04-eGFP:KanMX6 MATa, adel::HIS3, adel::KanMX6, HSP104-eGFP:KanMX6 MATa, lys2::HIS3, lys2::KanMX6, HSP104-eGFP:KanMX6 MATa, trp1::HIS3, trpl::KanMX6, HSP104-eGFP:KanMX6 MATa, cani::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMIX6, HSP104eGFP:KanMX6 MATa, intergenic region (119778-119573) between YHR0O6W and YHR0O7C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6 MATa, intergenic region (430900-431000) between YKL006C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKL0O6W::KanMX6, HSP104-eGFP:KanMX6 MATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, HSP104-eGFP:KanMX6 A31400 XI A31401 XII A31402 XIII A31403 XIV A31404 XV A31405 XVI A31406 - A31407 2n +11 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, HSP104-eGFP:KanMX6 MATa, adel6::HIS3, ade16::KanMX6, HSP104eGFP:KanMX6 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, HSP104-eGFP:KanMX6 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, HSP104-eGFP:KanMX6 MATa, leu9::HIS3, leu9::KanMX6, HSP104-eGFP:KanMX6 MATa, metl2::HIS3, met12::KanMX6, HSP104eGFP:KanMX6 MATa/a,MATa, adel::HIS3, lys2::KanMX6, HSP104eGFP:KanMX6 MATa/a, lys2::HIS3, lys2::KanMX6,lys2::LEU2, HSP104eGFP:KanMX6 98 MATa/cc, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, intergenic region (322250-322350) between YJL061W and YJL060W::LEU2, HSP104-eGFP:KanMX6 MATa/a, intergenic region (430900-431000) between YKL006C-A and YKL0O6W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, ntergenic region (430900-431000) between YKLO06C-A and YKLO06W::LEU2, HSP104eGFP:KanMX6 MATa/a, leu9::HIS3, leu9::KanMX6, leu9::LEU2,HSP104eGFP:KanMX6 MATa/a, HSP104-eGFP:KanMX6 MATa/a/a, HSP104-eGFP:KanMX6 MATa, hspl04::hspl04-eGFP-KAN adel::HIS3, lys2::KAN /YAC-6 MATa, hspl04::hsplO4-eGFP-KANadel::HIS3, lys2::KAN /YAC-3 MATa,, cdc28-4 A31408 2n+X A31409 2n+XI A31410 2n+XV A28220 A28219 - A28922 YAC-6 A28925 YAC-3 A29765 - A29766 - A25654 - MATa, Hspl04-eGFP::KanMX A32076 - MATa, adel::HIS3, lys2::KanMX6, pdr5::TRP1, YCP: A32077 I MATa, adel::HIS3, adel::KanMX6 pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 A32078 II A32079 IV A32080 - Hspl04-eGFP::KanMX V MATa,, cdc23-1, Hspl04-eGFP::KanMX pGAL-VHL-GFP:LEU2 MATa, lys2::HIS3, lys2::KanMX6 pdr5::TRP1, YCP: pGAL- VHL-GFP:LEU2 MATa, trpl::HIS3, trpl::KanMX6 pdr5::TRP1, YCP: pGAL- VHL-GFP:LEU2 MATa, canl::HIS3, intergenic region (187520-187620) between YERO15W and YER016W::KanMX6 pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 A32081 ViII MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6 pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 MATa, intergenic region (430900-431000) between A32082 IX YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, pdr5::TRP1. YCP: pGAL-VHLGFP:LEU2 MATa, intergenic region (322250-322350) between A32083 X 1_ YJL061W and YJL060W::HIS3, intergenic region (3222501 322350) between YJL061W and YJL060W::KanMX6 99 pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 A32084 XI MATa, intergenic region (430900-431000) between YKL006C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKL006C-A and YKL0O6W::KanMX6, pdr5::TRP1, YCP: pGAL-VHLGFP:LEU2 MATa, adel6::HIS3, adel6::KanMX6 pdr5::TRP1, YCP: A32085 XII A32086 XIII A32087 XIV A32088 XV A32089 XVI A30465 YAC-5 Mata, YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1, adel::HIS3, lys2::KAN/YAC3 A30467 YAC-6 Mata, YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1, pGAL-VHL-GFP:LEU2 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6 pdr5::TRP1, YCP: pGAL-VHLGFP:LEU2 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 MATa, leu9::HIS3, leu9::KanMX6 pdr5::TRP1, YCP: pGALVHL-GFP:LEU2 MATa, metl2::HIS3, metl2::KanMX6 pdr5::TRP1, YCP: pGAL-VHL-GFP:LEU2 adel::HIS3, lys2::KAN/YAC6 A30461 Mata, YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1, A30462 Mata, YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1, adel::HIS3, lys2::KAN, cdc23-1 MATa/a,YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1 MATa/a/a,YCP: pGAL-VHL-GFP:LEU2, pdr5::TRP1 MATa, adel::HIS3, lys2::KanMX6, pdr5::TRP1 MATa, adel::HIS3, adel::KanMX6, pdr5::TRP1 MATa, lys2::HIS3, lys2::KanMX6, pdr5::TRP1 MATa, trpl::HIS3, trpl::KanMX6, pdr5::TRP1 adel::HIS3, lys2::KAN, cdc28-4 A28388 A28389 A15549 A15551 Al 5553 Al 5555 I II IV Al 5557 vMATa, A15559 ViII canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, pdr5::TRP1 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6, pdr5::TRP1 A15561 IX MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, pdr5::TRP1 100 A15563 X A15566 XII A15567 xIII MATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, pdr5::TRP1 MATa, adel6::HIS3, adel6::KanMX6, pdr5::TRP1 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, pdr5::TRP1 Al 5569 XIV A15571 A15573 A32090 A32091 A32092 XV XVI A32093 V - I II MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNL0O4W::KanMX6, pdr5::TRP1 MATa, leu9::HIS3, leu9::KanMX6, pdr5::TRP1 MATa, met12::HIS3, met12::KanMX6, pdr5::TRP1 MATa, adel::HIS3, lys2::KanMX6, YCP: pGAL-csrc:URA3 MATa, adel::HIS3, adel::KanMX6, YCP: pGAL-csrc:URA3 MATa, lys2::HIS3, lys2::KanMX6, YCP: pGAL-csrc:URA3 MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, YCP: pGALcsrc:URA3 A32094 ViII MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, mtergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6, YCP: pGAL-csrc:URA3 MATa, intergenic region (430900-431000) between A32095 IX YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX, YCP: pGAL-csrc:URA36 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, YCP: pGAL-csrc:URA3 MATa, adel6::HIS3, adel6::KanMX6, YCP: pGAL- A32096 XI A32097 XII A32098 XIII A32099 XIV A32100 XV YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, YCP: pGAL-csrc:URA3 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, YCP: pGAL-csrc:URA3 MATa, leu9::HIS3, leu9::KanMX6, YCP: pGAL-csrc:URA3 A32101 XVI MATa, metl2::HIS3, metl2::KanMX6, YCP: pGAL- A32102 - MATa, adel::HIS3, lys2::KanMIX6, YCP: pGAL-vsrc:URA3 csrc:URA3 MATa, intergenic region (309200-309300) between csrc:URA3 101 A32103 A32104 I II A32105 V A32106 ViII A32107 IX A32108 XI A32109 XI A321 10 XIII A321 11 XIV A32112 XV A32113 XVI A31114 - A29843 I A29844 II A29845 V A29846 ViII A29847 IX MATa, adel::HIS3, adel::KanMX6, YCP: pGAL-vsrc:URA3 MATa, lys2::HIS3, lys2::KanM.X6, YCP: pGAL-vsrc:URA3 MATa, cani::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, YCP: pGALvsrc:URA3 MATa, intergenic region (119778-119573) between YHR0O6W and YHR007C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6, YCP: pGAL-vsrc:URA3 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX, YCP: pGAL-vsrc:URA36 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, YCP: pGAL-vsrc:URA3 MATa, adel6::HIS3, adel6::KanMX6, YCP: pGALvsrc:URA3 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, YCP: pGAL-vsrc:URA3 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, YCP: pGAL-vsrc:URA3 MATa, leu9::HIS3, leu9::KanMX6, YCP: pGAL-vsrc:URA3 MATa, netl2::HIS3, metl2::KanMX6, YCP: pGALvsrc:URA3 MATa, adel::HIS3, lys2::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, adel::HIS3, adel::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, lys2::HIS3, lys2::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, canl::HIS3, intergenic region (187520-187620) between YERO15W and YERO16W::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHR07C::KanMX6, , ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKL06W::KanMX6, , ura3:: stop2xEGFP::URA, 102 sup35::sup35-R2E2 X A29848 MIATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, , ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 XI A31110 MATa, intergenic region (430900-431000) between YKL0O6C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, , ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 A31 111 XII A29849 XIII MATa, adel6::HIS3, adel6::KanMX6, , ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 XCIV A31112 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6,, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 - kMATa, leu9::HIS3, leu9::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, metl2::HIS3, metl2::KanMX6, ura3:: stop2xEGFP::URA, sup35::sup35-R2E2 MATa, adel::HIS3, lys2::KanMX6, GAL-FLAG- A321 15 II MATa, lys2::HIS3, lys2::KanMX6, GAL-FLAGHttl(17AA)25QApro-CFP:URA3 A32116 V MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, GAL- A29450 XV A31113 XVI A32114 A32114 _ - Httl(17AA)25QApro-CFP:URA3 FLAG-Htt1(17AA)25QApro-CFP:URA3 A32117 VIII MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6 A32118 XI MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLOO6W::KanMX6, GAL-FLAG-Httl(17AA)25QAproCFP:URA3 A32119 XIII MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, GAL-FLAG-Httl(17AA)25QApro-CFP:URA3 103 A32120 XVI A32121 ~ A32122 1MATa, A32123 V A32124 VIII A32125 XI A32126 XIII A32127 XIIV A32128 XVI A32129 - A32130 II A32131 V A32132 ViII A32133 XI MATa, met12::HIS3, met12::KanMX6, GAL-FLAGHttl(17AA)25QApro-CFP:URA3 MATa, adel::HIS3, lys2::KanMX6, GAL-FLAGHttl(17AA)46QApro-CFP:URA3 lys2::HIS3, lys2::KanMX6, GAL-FLAGHttl(17AA)46QApro-CFP:URA3 MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, GALFLAG-Httl(17AA)46QApro-CFP:URA3 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, GAL-FLAG-Httl(17AA)46QAproCFP:URA3 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, GAL-FLAG-Httl(17AA)46QAproCFP:URA3 MATa, intergenic region (622880-622980) between YNLO05C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNLO04W::KanMX6, GAL-FLAG-Httl (1 7AA)46QApro-CFP:URA3 MATa, metl2::HIS3, met12::KanMX6, GAL-FLAGHttl(17AA)46QApro-CFP:URA3 MATa, adel::HIS3, lys2::KanMX6, GAL-FLAGHttl(17AA)72QApro-CFP:URA3 MIATa, lys2::HIS3, lys2::KanMIX6, GAL-FLAGHttl (17AA)72QApro-CFP:URA3 MIATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, GALFLAG-Httl(17AA)72QApro-CFP:URA3 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6 GAL-FLAG-Htt (17AA)72QApro-CFP:URA3 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, GAL-FLAG-Httl(17AA)72QAproCFP:URA3 104 A32134 XIII A32135 XVI A32407 A32408 II A32409 V A32410 ViII A32411 X A32412 XI A32413 XIII A32414 A32415 A32416 XV XVI A32417 V A32418 ViII A32419 X A32420 XIII A32421 XV - MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, GAL-FLAG-Httl(17AA)72QAproCFP:URA3 MATa, metl2::HIS3, met12::KanMX6, GAL-FLAGHttl(17AA)72QApro-CFP:URA3 MATa, adel::HIS3, lys2::KanMX6, SSA1-3HA:TRP1 MATa, lys2::HIS3, lys2::KanMX6 SSA1-3HA:TRP1 MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, SSA13HA:TRP1 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHRO07C::KanMX6, SSA1-3HA:TRP1 MATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, SSA13HA:TRP1 MATa, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKLO06W::KanMX6, SSA1-3HA:TRP1 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, SSA1-3HA:TRP1 MATa, leu9::HIS3, leu9::KanMX6, SSA1-3HA:TRP1 MATa, metl2::HIS3, metl2::KanMX6, SSA1-3HA:TRP1 MATa, adel::HIS3, lys2::KanMX6, SSE2-3HA:TRP1 MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6,SSE23HA:TRP1 MATa, intergenic region (119778-119573) between YHRO06W and YHRO07C::HIS3, intergenic region (119778119573) between YHRO06W and YHR07C::KanMX6,SSE2-3HA:TRP1 MATa, intergenic region (322250-322350) between YJL061W and YJL060W::HIS3, intergenic region (322250322350) between YJL061W and YJL060W::KanMX6, SSE23HA:TRP1 MATa, intergenic region (309200-309300) between YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, MATa, leu9::HIS3, leu9::KanMX6, SSE2-3HA:TRP1 105 A32422 A32423 A32424 XVI II A32425 ViII MATa, metl2::HIS3, met12::KanMX6, SSE2-3HA:TRP1 MATa, adel::HIS3, lys2::KanMX6, HSP42-3HA:TRP1 MATa, lys2::HIS3, lys2::KanMX6, HSP42-3HA:TRP1 MATa, intergenic region (119778-119573) between YHRO06W and YHR007C::HIS3, intergenic region (119778119573) between YHR0O6W and YHR0O7C::KanMX6,HSP42-3HA:TRP1 MATa, intergenic region (309200-309300) between A32426 XIII A32427 XIV A32428 XVI A33369 - YMR017W and YMR018W::HIS3, intergenic region (309200-309300) between YMR017W and YMR018W::KanMX6, HSP42-3HA:TRP1 MATa, intergenic region (622880-622980) between YNL0O5C and YNLO04W::HIS3, intergenic region (622880622980) between YNLO05C and YNL0O4W::KanMX6, HSP42-3H-A:TRP1 MATa, metl2::HIS3, met12::KanMX6, HSP42-3HA:TRP1 MATa, adel::HIS3, lys2::KanMX6, ubp6::TRP1, HSP104- A33370 II MATa, lys2::HIS3, lys2::KanMIX6, ubp6::TRP1, HSP104- V MATa, canl::HIS3, intergenic region (187520-187620) between YER015W and YER016W::KanMX6, ubp6::TRP1, A33371 eGFP:KanMX6 eGFP:KanMX6 HSP104-eGFP:KanMX6 MATa, intergenic region (430900-431000) between A33372 IX YKLO06C-A and YKLO06W::HIS3, intergenic region (430900-431000) between YKLO06C-A and YKL06W::KanMX6,ubp6::TRP1, HSP104-eGFP:KanMX6 106 References Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P et al. 2004. 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Yurov YB, Vorsanova SG, Iourov IY, Demidova IA, Beresheva AK, Kravetz VS, Monakhov VV, Kolotii AD, Voinova-Ulas VY, Gorbachevskaya NL. 2007b. Unexplained autism is frequently associated with low-level mosaic aneuploidy. J Med Genet 44: 521-525. Zhu J, Pavelka N, Bradford WD, Rancati G, Li R. 2012. Karyotypic determinants of chromosome instability in aneuploid budding yeast. PLoSgenetics 8: e1002719. 110 Chapter 3: Conclusions and Future Directions 111 Summary of key conclusions The study of aneuploidy has advanced tremendously in the last few years, in large part due to systematic and rigorous analyses of aneuploid cells with diverse karyotypes. The ability to generate a diverse panel of aneuploid Saccharoyces cerevisiae strains (both stable aneuploid strains of a defined karyotype and highly aneuploid albeit genomically unstable strains) and the many tools that have been developed to study cellular biology in this model organism have, in no doubt, enabled the field to pursue questions that had previously never been addressed. Using this approach, we have been able to separate the phenotypes that are consequences of specific karyotypic imbalances, i.e. due to the karyotype itself and those that are general consequences of harboring an imbalanced genome, irrespective of which chromosomes are in excess. The research in this thesis expands upon our knowledge of how aneuploidy interacts with, and the consequences it has on the basic cellular process of protein homeostasis. Understanding the relationship between the maintenance of genomic integrity and proteostasis has provided us with great insight that may, in part, explain why aneuploidy has such striking effects on cellular fitness. Since protein homeostasis is a dilemma that is encountered by all cells and the regulatory programs that govern it are well conserved, the research discussed in this thesis may shed light on the effects of aneuploidy in higher eukaryotes including humans. Furthermore, understanding the consequences of aneuploidy on cellular physiology, and in particular on proteostasis could have a profound effect on our view and treatment of human pathologies that harbor unbalanced karyotypes such as Down's Syndrome and most solid tumors. Protein production is carefully controlled in all cells. As this process requires an extraordinary amount of energy and resources for both translating the peptide and folding it, the cell rarely produces superfluous protein molecules. The protein quality control 112 machinery (chaperones and proteasome) are well calibrated to ensure protein homeostasis in normal growth conditions and there are transcriptional programs that upregulate the cell's quality control capacity that are initiated in conditions of acute proteotoxic stress. The amount of protein molecules produced is regulated both at the transcription and translation levels, and thus protein subunits that coalesce into protein complexes are generated at stoichiometric levels. That is to say that if protein A binds forms a heterotrimeric complex with two molecules of protein B, there will be approximately twice as many molecules of protein B than of protein A in the cell. As I showed in Chapter 2, aneuploidy poses a severe problem for protein homeostasis. The chromosomes in excess are, for the most part, translated into protein that must then be folded and degraded by the quality control machinery. Not only does the cell have to cope with excess protein molecules, it must tackle the stoichiometric imbalances that are generated when complex subunits are expressed on chromosomes that are present in different copy numbers. Aneuploidy results in the formation of protein aggregates and we speculate that this is due to the increased folding burden on the quality control machinery. I showed this effect specifically on the ubiquitous chaperone Hsp90 and we determine that aneuploidy appears to exhaust Hsp90's folding capacity. Furthermore, aneuploidy sensitizes cells to the detrimental effects of toxic hard-tofold proteins associated with human neurodegenerative disease suggesting that this phenomenon may have implications beyond the study of cancer and developmental disease. In this section, I will discuss several issues that arise from this research, centering the discussion on 4 central questions: (1) How does aneuploidy exhaust the cell's folding capacity? (2) What proteins comprise the protein aggregates found in aneuploid strains? (3) How is the mammalian proteome affected by aneuploidy? (4) Is there a relationship between aneuploidy, aging and neurodegenerative disease? 113 Aneuploidy exhausts the cell's protein quality control capacity Why are aneuploid cells aggregate-prone? In Chapter 2, I described how the alterations in the proteome brought along by aneuploidy result in proteotoxic stress and lead to an accumulation of protein aggregates. Aggregates could be a result of overwhelmed folding pathways or could stem from reduced chaperone activity. . In vitro activity studies are needed to determine if the folding activity of chaperones purified from aneuploid strains is significantly different than that of euploid yeast strains. Both alternatives are possible, but given that chaperone activity is crucial to the viability of the cell and that the aneuploid chromosomes are actively expressed, it is likely that excess proteins produced from the aneuploid chromosomes occupy chaperones and thereby reduce their availability to assist in the folding of their other clients What deternines the extent of aggregate formation in aneuploid cells is not yet known. It does not appear to correlate with either the degree of aneuploidy (by DNA content), total protein in excess, delay in G1, or proliferation rate. However, it is important to bear in mind that our aggregate analysis by HSP104-eGFPfoci does not measure absolute amounts of aggregated proteins in cells and, more importantly, is not able to distinguish toxic from nontoxic aggregates. It is interesting to note that the Environmental Stress Response (ESR) (Gasch et al. 2000), which encompasses part of the heat- shock response, correlates with degree of aneuploidy (Torres et al. 2007, Sheltzer, 2012 #4286). I propose that protein aggregate burden correlates with the number of obligate chaperone clients and hence with the distribution of their encoding genes in the genome. This hypothesis has proven difficult to test as our understanding of client specificity for many chaperones is poor and we do not yet have a comprehensive list of obligate chaperone clients. Additionally, chaperones have redundant functions and 114 although a client may generally have a defined folding pathway, it will get folded by alternative means if those chaperones are non-functional or overwhelmed (Hard et al. 2011). It may seem surprising that the cells cannot maintain proteostasis under the presence of a single additional chromosome, which, depending on chromosome size, results in 20 /- 12% of the genome being imbalanced. Previous studies showed that even small amounts of non-functional, seemingly non-toxic, misfolded proteins (Ura3, YFP) place a burden on the cell's protein quality-control systems and hence adversely affect cellular fitness. Expression of a single misfolded cytosolic protein at <0.1% of total protein leads to a significant decrease in proliferative abilities and the induction of a cytoplasmic unfolded protein response (Geiler-Samerotte et al. 2011). As aneuploid cells express full chromosomes that are comprised of proteins that are functional, it is not surprising that even a 2% increase in protein production placed on the protein quality control machinery is great could be detrimental especially when consider the overproduction of proteins encoded on the extra chromosomes that require chaperones for their function, such as protein kinases and WD40 repeat proteins. It is important to keep in mind that excess protein molecules is not the only proteomic alteration that aneuploid cells must tolerate. It is well established that many subunits of protein complexes only acquire a stable conformation by binding to other subunits of the complex (Imai et al. 2003; Boulon et al. 2008). Thus, every single polypeptide produced by genes located on aneuploid chromosomes that normally has a binding partner is-in the disomes-in excess. Every single subunit that requires a chaperone to maintain solubility until it can bind to its binding partner (that, in case of aneuploid cells, does not exist) will occupy the chaperone indefinitely until it is degraded. I pose forth a model in which it is this dramatic change in protein stoichiometries that leads to an increased burden 115 on the protein quality-control pathways of the cell (Figure 1). Individual subunits present in excess require the continuous assistance of chaperones, titrating chaperones away from assisting other folding reactions and reducing the general folding capacity of the cell, and thus interfering with their essential function of mediating folding of essential proteins (Hard et al. 2011). This model is supported by my finding that the aggregate phenotype was ameliorated when the ratio of uncomplexed proteins to properly complexed proteins was decreased by increasing base ploidy (as in trisomic 2n+1 strains, see Figure 1). This data strongly suggests that the proteotoxicity observed in aneuploids is indeed in part the result of the protein stoichiometry imbalances caused by aneuploidy, although it is also possible that diploid cells are more efficient at clearing aggregates. In summary, aneuploidy impacts protein homeostasis in multiple ways so that even small un-balanced chromosomes have a significant impact on the cell's protein quality-control systems. 116 FIGURE 1 Stoichiometric imbalance increases the burden on quality control pathways. Aneuploidy causes stoichiometric imbalances, resulting in protein complex subunits that do not have binding partners available. Partially folded subunits (represented in blue here) often bind to chaperones to maintain stability while they await a binding partner, but if their partner is non-existent, they will be bound to chaperones until they are degraded. When the basal ploidy is increased (from n to 2n in this example) but the number of excess chromosomes remains the same, the relative imbalance is reduced and the burden on the cell's quality control machinery is also reduced. Figure 1 Haploid (n) cell Clients Protein complex DNA A Aneuploid (n+1) cell OW qW VW 1W vw W 4M Ift 1 1"W IM Diploid (2n) cell Aneuploid (2n+1) cell 1 / 5/ Q.CtrI. capacity 2/ I. I f 4f 6/2 4 JO -U 117 The folding capacity of chaperonesis alteredby genomic imbalances In this thesis, I have shown that aneuploidy results in dramatic consequences for the quality control machinery of the cell. Using assays that monitor Hsp90 activity in vivo I showed that 8 out of 11 disomes (haploid +1 chromosome) tested showed reduced Hsp90 folding activity (Oromendia et al. 2012). This was surprising given that Hsp90 has been shown to be highly abundant in yeast, representing 1-2% of total protein (Borkovich et al. 1989; Neckers 2007). It is not yet known why Hsp90 activity is limiting in so many different disomic yeast strains. I can envision two possible scenarios: either aneuploidy affects the chaperone's intrinsic catalytic activity and causes a reduction in folding capacity or the presence of excess client proteins brought upon by aneuploidy depletes the Hsp90 reservoir. In vitro folding assays are needed to exclude the possibility of a reduction in the folding activity of the Hsp90 molecules but it would be surprising if 8 different aneuploidies caused an intrinsic defect in a chaperone as essential as Hsp90. Hsp90 is unlikely to the only chaperone system limiting in aneuploid yeast strains. I hypothesize that, when analyzed, most chaperone families will be limiting in at least some aneuploid strains. As with Hsp90, it would be of special interest to perform in vivo or in whole extract chaperone assays to be able to assess whether the folding capacity (as opposed to inherent biochemical activity) is impaired. In vivo/in extract protein folding assays have been developed for numerous chaperone families: to assay TRiC/CCT activity in vivo one can measure the relative ratio of native and misfolded actin (native actin binds with high affinity to DNAseI coated beads). De novo folding of luciferase, and thus its activity, requires Hsp70; by measuring luminescence after expression of luxAB, one can determine the in vivo folding capacity of Hsp70. To measure the disaggregation capacity of Hsp104, one can express luciferase and heat shock cells. The heat shock will cause aggregation of 118 luciferase and loss of luminescence, the recovery of luciferase activity (and luminescence) is directly dependent on Hsp104. Performing these assays on a panel of aneuploid strains will enable us to generate a data set that analyzes the activities of various chaperones when different proteins are present in excess. Different aneuploidies will, of course, affect the various chaperone families differently depending on the proportion of clients for a particular chaperone affected by a given aneuploidy. A neuploidy is a chronic stress, distinctform environmentalproteotoxicstressors The proteotoxic stress that aneuploidy brings upon cells is distinct from environmental proteotoxic stressors. An environmental stressor such as high heat, or sharp transitions into osmotic stress usually brings upon a sharp transition between a state of proteostasis and one of severely altered and mis-folded proteins. For example, a 30 minute exposure to 42C results in misfolding of over 50% of the yeast proteome (Richter et al. 2010). This abrupt change results in an immediate need for increased quality control capacity relieved by the transcriptional up-regulation of chaperones and down-regulation of protein synthesis that comprise the heat-shock response and similar responses in other organelles (UPR, mitoUPR). These responses are costly to the cell, not only in chaperone production and function but also in the lack of biomass produced while translation is dampened. As environmental stressors are most often severe but transient, the transcriptional responses are tailored to resolve proteotoxic stress and rapidly return to basal level so the cell can continue with its normal function. As such, the heat shock response and other similar responses are transient transcriptional programs tailored for resolving acute proteotoxic stress. In Chapter 2, we show that although, when prompted, disomic yeast are capable of mounting both the heat shock and unfolded protein response with similar kinetics as wild119 type cells they appear to be unable to maintain protein homeostasis under normal growth conditions. This is not surprising as aneuploidy results in a mild, but persistent proteotoxic stress. Saccharoyces cerevisiae cells that are exposed to a severe heat shock (42*C for 15 min) accumulate large amounts of protein aggregates which can be quantified both by the percentage of cells that harbor HSP104-eGFP aggregates (100% of cells contain many large eGFP foci) or via a simple fractionation and visualization of total protein in the pellet fraction on an SDS-PAGE gel stained for total protein with Coomassie stain (Figure 2). In contrast, both the percentage of cells that contain HSP104-eCFP aggregates (Oromendia, 2012) and the accumulation of proteins in the pellet fraction are far less severe in aneuploid cells. Whereas environmental proteotoxic stressors result in transient but dramatic transformations of the proteome, aneuploidy leads to persistent but relatively mild alterations in protein homeostasis. We believe that it is this difference that prevents the canonical transcriptional responses to protein misfolding (HSR, UPR) from being evoked. The contrast between the proteotoxic stress caused by environmental insults and that caused by aneuploidy prompts us to question whether general upregulation of protein quality control machinery would be sufficient to ameliorate proteotoxicity in aneuploid yeast and, perhaps, result in an improvement in proliferation rates. Torres et al. (Torres et al. 2007)have previously shown that enhancing the degradation of highly abundant proteins by the proteasome by deleting the ubiquitin ligase UBP6 is sufficient to improve growth rates in a subset of disomes. We later showed that this deletion could ameliorate, but not fully suppress the accumulation of protein aggregates in disomic yeast (Oromendia, 2012). In future work, it will be of interest to assess the effects of enhancing the quality control capacity of the cell by increasing the abundance or activity of protein chaperones on aneuploid cells. This could be achieved by simply increasing the abundance of one, or a 120 subset of protein chaperones by increasing the copy number or placing them under the control of a strong, constitutive promoter but as many chaperones function in large complexes with many co-chaperones and accessory proteins an up-regulation of a subset of chaperones may not be sufficient to improve protein quality control. An alternative approach is to artificially induce a heat shock response in aneuploid cells; this would result of up-regulation of a suite of chaperones and cofactors that are already poised to rescue cells from proteotoxic distress. The master regulator of the heat shock response in S. cerevisiae is the transcription factor Hsfl. Hyperphosphorylation and the resulting activation of Hsf1 results in the transcription of a large suite of genes involved in protein folding, carbohydrate metabolism, and energy generation (reviewed in (Richter et al. 2010). Both the hyperactive allele of HSFJ, hsf1147-833 (Sorger 1990) and the overexpression of an upstream regulator GIP2 (Yeger-Lotem et al. 2009) have been shown to cause overexpression of HSF1 targets. Generating aneuploid strains that contain these alleles and assessing whether an artificially induced heat shock response reduces the percentage of cells harboring protein aggregates or improves proliferative capacity would enable us to determine whether increasing protein quality control capacity is sufficient to counteract the detrimental effects that aneuploidy has on the proteome. Additionally, this would suggest that aneuploidy is simply over-burdening the quality control machinery and titrating away chaperones from proteins that require them. As mentioned before, the canonical responses to protein misfolding are energetically costly and thus one major caveat of this reasoning is that, if increasing quality control capacity in the cell by over-expressing chaperones or artificially inducing a heat shock response is too taxing and requires too much of the cells' energy stores we may not be able to observe an improvement in proliferation nor a reduction in protein aggregate accumulation. 121 The composition of protein aggregates in aneuploid yeast Aneuploidy results in the accumulation of protein aggregates under conditions of normal, non-stress growth conditions (Chapter 2) and this phenotype is exacerbated under conditions of mild proteotoxic stress. This finding brings about an important issue that is yet unresolved: which proteins comprise the protein aggregates and is there a reason why they end their life in protein aggregates? Two non-exclusive models could explain the identity of the proteins that result in the protein aggregates in aneuploid cells: the aggregates in each aneuploid strain could be comprised mainly of proteins that are encoded by the chromosome in excess or, the aggregates found in all strains could have similar composition irrespective of the karyotype of the cell. In the latter case, the aggregates could be formed mainly by proteins that are especially difficult to fold and that are obligate clients of chaperones and when these are over-burdened they do not manage to acquire native structure and terminate as aggregated folding intermediates. I have now managed to develop an aggregate purification protocol that is both reproducible and comprehensive and that shows a clear differential between wild type and disomic strains (Figure 2). Using this purification method, identifying the proteins that make up the protein aggregates observed in aneuploid cells is now achievable via quantitative mass spectrometry. Using the Stable Isotope Labeling by Amino acids in Cell Culture (SILAC) technique one can purify aggregates from differentially labeled wild type and aneuploid cultures and after mass spectrometry determine quantitatively which proteins comprise the aggregate fraction of the cell. Once one has done this with a panel of different aneuploidies that each have a different chromosome in excess, one can use bioinformatics to analyze the datasets and determine whether they are more similar to each other (the identity of the 122 proteins that aggregate is irrespective of the karyotype) or they are more similar to the proteins encoded by the particular chromosome present in additional copies (suggesting that most proteins that terminate in aggregates were proteins that were in excess). Elucidating the identity of the proteins that conform the protein aggregates in aneuploid yeast may glean insight on how the cell determines which proteins will be aggregated. If the protein composition of aggregates is different according to karyotype and the proteins that aggregate are those that are present in excess because of the presence of an additional copy of the chromosome that encodes for them, one can imagine two nonexclusive mechanisms via which they could terminate as aggregated forms. Given that they are overabundant, proteins present in excess might be, by sheer stochasticity, more likely to end up in protein aggregates, especially if they are highly abundant proteins in their natural copy number state. 123 FIGURE 2- Purification of protein aggregates from Aneuploid yeast. Protein aggregates were purified from a haploid yeast strain (WT) and strains disomic for chromosome II and chromosome VIII carrying an Hspl04-GFP fusion protein. As a positive control we used wild type cells grown at 25C and heat-shocked for 30 minutes at 42'C (A). Cells were grown in rich medium (YPD) at 25 0 C (B), 30 0 C (C) and 34 0 C (D) to OD6 0 1 and 25 mL were collected for fractionation. Cells were incubated for 20 min at 25*C in Lysis buffer (20mM NaPI pH6.8, 10mM DTT, 1mM EDTA, 0.1% Tween, 1x Roche Protease Inhibitor, 1mM PMSF and 2.5mg/ml 20T Zymolyase) and then lysed by sonication (two rounds of 8x, level 4, 50% on a Branson Sonifier). The lysate was spun for 20 min at 1600rpm and the protein concentration was equalized to 3mg/ml (Total Protein Sample, T). The aggregates were then fractionated by spinning for 20 min 16000xg (Supernatant Sample, S), washing twice with 2% NP40 in 20 mM NaPI, 1x protease inhibitor, 1mM PMSF, sonicating 6x, level 4 50% duty cycle in between washes. . The pellet fraction was spun again and washed with 20 mM NaPi, 1x protease inhibitors, 1 mM PMSF and sonicated 4x, level 2, 65% duty cycle and spun a final time at 16 0 0 0 xg for 20 min. The pellet fraction (P) was resuspended in 8M urea. Total, Supernatant and Pellet fractions were run on an SDS-PAGE gel and stained with Coomassie. We also performed an anti-GFP western blot to detect Hspl04-GFP 124 Figure 2 A 0 YPD 25 C 30min 420C B Coomassie a-GFP T S P T S P YPD 250C YPD 250C Pellet Sup. Total WT 11 VIll W1 11Vill WT 11 Vill Total Sup. Pellet WT 11 Vill WT 11 Vill WT II Vill Iw YPD 300C YPD 300C Pellet Sup. Total WT 1I Vill WT 11 Vill WT 11 Vill Sup. Pellet Total WT iI Vill WT iI Vill WT iI Vill C LL. Asia D YPD 34*C YPD 340C Pellet Sup. Total WT 11 Vill WT 11 Vill WT 11 Vill Pellet Sup. Total WT 11 VIII WT it Vill WT II Vill Apof a. LL Irv Wop 40 125 Alternatively, proteins that are overabundant may be actively partitioned into aggregates as means to reduce aberrant interactions that may cause toxicity and to decrease burden on the quality control pathways of the cell. Although protein aggregates have long been thought of as toxic species that hamper cellular function, reports in the literature suggests that they may, at least in cases of protein folding diseases, be cytoprotectant. Perhaps aneuploid cells recognize that there is an excess of a subset of proteins (because their coding sequence is present in excess) and there is an active process that attempts to sequester these protein subunits to limit the damage they can cause. This would require an active sorting process where the cell would be able to recognize that there are protein units that are not being utilized and then selectively target those for aggregation and presumably later degradation. While there have been no post-translational modifications described that specifically target proteins for aggregation, the addition of ubiquitin chains to proteins has been shown to mark them for protein degradation by the UPS system. One can take advantage of the di-Gly remnant after trypsinization of the isopeptide bond formed between the Lysine on the target protein and the C-terminal Glycine of ubiquitin to immunopurify and identify via mass spectrometry those proteins that were ubiquitinated when the sample was collected (Kim et al. 2011). Using di-Gly mass spectrometry one could determine if there is an overrepresentation of proteins encoded by the extra chromosome in the subset of ubiquitinated proteins, which could suggest pre-emptive targeting for degradation previous to protein misfolding. In an alternative model where the composition of protein aggregates is irrespective of the genomic imbalance present, the determinant for which proteins constitute the protein aggregates found in aneuploid strains could be the difficulty that they have in acquiring a stable, soluble native structure. Limited protein folding capacity would result in a higher 126 proportion of those more 'demanding' proteins to remain unfolded/misfolded and a higher proportion of the more easily folded ones to remain soluble. As this scenario would not require the cell to sort its proteins into those that are present in excess and those that are not, I believe it to be more likely. Purification and identification of the protein aggregate constituents will inform us whether either one, or both of these models is accurate. Aneuploidy in mammalian cells alters protein quality control As I have shown in Chapeter 2, aneuploidy dramatically alters protein quality control in Saccharomyces cerevisiae- there is a pressing need in the field to determine if the same is true in aneuploid mammalian cells. Similarly to aneuploid S. cerevisiae (Torres et al. 2010), aneuploid human cell lines created by chromosome transfer also appear to fully transcribe the tetrasomic chromosomes (average mRNA aneuploid to diploid log2 ratio of the extra chromosome is 1.09) and to, at least partially translate it (average protein aneuploid to diploid ratio of proteins encoded by the tetrasomic chromosome is 0.69) (Stingele et al. 2012). Interestingly, as in aneuploid yeast cells, there appears to be a subset of proteins (between 20 and 25% in both human and yeast), that are retained at disomic levels; as their mRNA is present at copy number levels, it appears that they're expression is controlled at the protein level (Torres et al. 2010) (Stingele et al. 2012). The vast majority of proteins that are not present in levels reflecting the increased copy number are members of protein complexes. The mechanism via which this translational control occurs has not yet been determined, but it is an active area of research in the field. Although there have not been any reports of protein aggregation in mammalian aneuploid cells, recent work from our lab and others has suggested that aneuploidy could also be disturbing protein homeostasis in mammalian cells both in similar and different ways 127 than it is altered in yeast. Aneuploidy in all species analyzed shows a common transcriptional response reminiscent of the ESR (Sheltzer et al. 2012), this response includes upregulation of a subset of protein chaperones. In aneuploid yeast, this transcriptional up regulation of chaperones does not result in a higher protein abundance of any of the chaperones tested as a general response to aneuploidy (Chapter 2). Although this is also true for most chaperones tested in trisomic mouse embryonic fibroblasts (MEFs) trisomic for chromosomes 13, 16 or 19, all trisomic MEFs harbor consistently higher abundance of the inducible isoform of Hsp70: Hsp72 (Tang et al. 2011). We have also determined that trisomic MEFs appear to show an altered heat shock response, with many genes upregulated to a higher degree than wild type (Y.C Tang and S. Pfau, unpublished results). It remains to be seen if this is due to differential kinetics of the heat shock response or if the response maintains its kinetics but its intensity increased to aid in clearance excess misfolded proteins. I, and others have shown that aneuploidy sensitizes yeast cells to proteasome inhibitors and that increasing proteasme activity partially ameliorates the protein aggregation phenotypes (Chapter 2, (Torres et al. 2007)). In contrast, trisomic MEFs have not been found to be sensitive to the proteasome inhibitor Bortezamid, suggesting that mammalian aneuploidy does not add stress to the proteasome system (YC Tang, unpublished observations). This is unsurprising as rather than relying on the proteasome for protein degradation, mammalian cells deploy autophagy as a major means to deal with aggregated proteins (Tyedmers et al. 2010). In fact, mouse and human aneuploid cells were shown to have increased levels of the autophagosome marker LC3-II (Stingele et al. 2013). Similar to yeast, aneuploid cells are unable to maintain quality control- in mammalian cells this is observed as an accumulation of autophagosomes in the lysosome. (S. Santaguida, unpublished results). 128 It is clear that both mammalian and yeast aneuploid cells are under proteotoxic stress, but the pathways that are affected appear to differ. Further studies characterizing the role of protein chaperones in mammalian aneuploidy would provide a more comprehensive picture of the general effects of aneuploidy on eukaryotic cells. It would also be exciting to hyperactivate the heatshock response in either aneuploid MEFs or human lines and assess the effects of increasing quality control capacity on the proliferation of these cells. Interface between aneuploidy, aging and neurodegeneration Recent work has described a relationship between karyotypic imbalances and aging. Mice that carry a hypomorphic allele of the spindle assembly checkpoint protein BUBRI and thus missegregate chromosomes readily show signs of progeria (premature aging) (Baker et al. 2004; Wijshake et al. 2012; Baker et al. 2013b). Conversely, mice that overexpress BUBR1 show reduced chromosome mis-segregation rates and extended lifespans (Baker et al. 2013a). These data strongly suggest a relationship between the process of aging and of aneuploidy; the nature of this relationship is still poorly understood. It is interesting to note that one of the defining characteristics of an aging cell is the breakdown of protein homeostasis and the accumulation of protein aggregates (Lopez-Otin et al. 2013), as this is the same phenotype we observe in aneuploid cells it is tempting to posit whether the breakdown in protein quality control is causal to aging. If so, premature aging seen in the hypomorphic BUBR1 mice could be the result of the proteotoxic stress and resulting breakdown in protein quality control caused by aneuploidy. In depth studies of the quality control capacity of cells carrying a hypomorphic allele of BUBR1 are needed to start assessing the validity of this model. As hypomorphic BUBR1 mice are also the mouse model for Mosaic Aneuploid Variegated (MVA) Syndrome, it would be of interest to assess the protein quality control 129 capacity in cultured cells derived from MVA patients. MVA is a pediatric syndrome characterized by the early onset of tumors, but it can also be described as a progeria syndrome as patients show symptoms such as growth retardation and cataracts that are reminiscent of those seen in premature aging disorders. Although reduced lifespan has been shown for mice carry hypomorphic BUBR1 alleles, a causal relationship between chromosome mis-segregation or aneuploidy and aging of cells has yet to be described. There are no other reports of mouse models of chromosome mis-segregation leading to changes in lifespan but most studies did not have as a goal to determine aneuploidy's effect on lifespan and mice were sacrificed in their youth. More detailed studies with the goal of unveiling any possible relationship between chromosome mis-segregation and aging need to be done to elucidate the potential relationship between them. Age has been shown to be the most predictive and predisposing factor for common neurodegenerative ailments such as Alzheimer's, Parkinson's and Lou Gherig's disease. These diseases all share similar etiologies: a misfolded protein that wreaks havoc on cellular homeostasis causing neuronal death. It is unclear why age is such a preponderant predisposing factor, but it is believed that the breakdown in protein quality control and accumulation of protein aggregates in aged cells may lead to a reduction in quality control capacity and to the misfolding and formation of toxic disease proteins. Concluding Remarks Aneuploidy severely impacts cellular physiology, affecting almost every cellular process. In this thesis I have shown that proteomic alterations and imbalances caused by aneuploidy negatively impact the cell's ability to maintain protein homeostasis. I have elucidated the impact of aneuploidy of the ubiquitous chaperone Hsp90 and determined that 130 aneuploidy sensitizes cells to situations that demand high folding capacity. Together, the data in this thesis demonstrate that, in addition to gene specific effects, a state of chromosomal imbalance has dramatic consequences for the cellular protein quality control pathways. Much remains to be learned, especially in understanding the mechanisims by which aneuploidy disturbs protein homeostasis and the cellular attempts to ameliorate this disturbance. It will be important to glean molecular insight and to understand which quality control systems, if any, are more severely affected and determine if there are ways to ameliorate these defects. It is my hope that with further mechanistic understanding of this process, ongoing research will contribute to our understanding, and potentially to the treatment of disease conditions for which aneuploidy is a central part of their etiology, be they cancer or developmental syndromes. 131 References Baker DJ, Dawlaty MM, Wijshake T, Jeganathan KB, Malureanu L, van Ree JH, CrespoDiaz R, Reyes S, Seaburg L, Shapiro V et al. 2013a. Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan. Nature cell biology 15: 96-102. -. 2013b. 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