Controlling Protein Homeostasis Through Regulation of Heat Shock Factor 1 Anna E

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Controlling protein homeostasis through regulation of Heat shock factor 1 Anna E. Masser Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Tuesday 21 May 2019 at 10.00 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20.

Abstract In order to thrive in a changing environment all organisms need to ensure protein homeostasis (proteostasis). Proteostasis is ensured by the proteostasis system that monitors the folding status of the proteome and regulates cell physiology and gene expression to counteract any perturbations. An increased burden on the proteostasis system activates Heat shock factor 1 (Hsf1) to induce transcription of the heat shock response (HSR), a transiently induced transcriptional program including core proteostasis genes, importantly those encoding the Hsp70 class of molecular chaperones. The HSR assists cells in counteracting the harmful effects of protein folding stress and restoring proteostasis. The work presented in this thesis is based on experiments with the Saccharomyces cerevisiae (yeast) model with the overall goal of deciphering how Hsp70 detects and impacts on perturbations of cellular proteostasis and controls Hsf1 activity. In Study I we describe the fundamental mechanism by which Hsp70 maintains Hsf1 in its latent state by controlling its ability to bind DNA. We found that Hsf1 and unfolded proteins directly compete for binding to the Hsp70 substrate-binding domain. During heat shock the pool of unfolded proteins mainly consist of misfolded, newly synthesized proteins. Severe out-titration of Hsp70 by misfolded substrates resulted in unrestrained Hsf1 activity inducing a previously uncharacterized genetic hyper-stress program. More insight into regulation of Hsp70 availability was gained in Study II where the two splice isoforms of the Hsp70 nucleotide exchange factor Fes1 were characterized. We found that the cytosolic splice isoform Fes1S is crucial to release unfolded proteins from Hsp70 and that impaired release results in strong Hsf1 activation. In Study III we developed methodology to easily measure the rapid changes in Hsf1 activity upon proteostatic perturbations and to monitor protein turnover using the novel bioluminescent reporter NanoLuc optimized for yeast expression (yNluc). In Study IV we report that yNluc also functions as an in vivo reporter that detects severe perturbations of de novo protein folding by its failure to fold to an active conformation under such conditions. Finally, in Study V we investigated how organellar proteostasis impacts on the availability of cytosolic Hsp70. We found that a lowered mitochondrial proteostatic load as a result of high translation accuracy extended lifespan and improved cytosolic proteostasis capacity, evidenced by more rapid stress recovery and less sensitivity to toxic misfolded proteins. In contrast, lowered mitochondrial translation accuracy decreased lifespan and impaired management of cytosolic protein aggregates as well as elicited a general transcriptional stress response. Taken together, the findings presented in this thesis advance our understanding of how the regulatory mechanisms of the proteostasis system function. Furthermore, they provide novel methodology that will facilitate future studies to improve our understanding how cells integrate internal and external stress cues to control proteostasis.

Keywords: stress, protein homeostasis, molecular chaperones, heat shock response, Hsf1, Hsp70, Saccharomyces cerevisiae.

Stockholm 2019 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-167081

ISBN 978-91-7797-688-2 ISBN 978-91-7797-708-7

Department of Molecular Biosciences, The Wenner-Gren Institute

Stockholm University, 106 91 Stockholm

CONTROLLING PROTEIN HOMEOSTASIS THROUGH REGULATION OF HEAT SHOCK FACTOR 1

Anna E. Masser

Controlling protein homeostasis through regulation of Heat shock factor 1 Anna E. Masser ©Anna E. Masser, Stockholm University 2019

ISBN print 978-91-7797-688-2 ISBN PDF 978-91-7797-708-7

Cover illustration by Anna E. Masser

Back cover photography by Deike J. Omnus

Printed in Sweden by Universitetsservice US-AB, Stockholm 2019 Till min familj

RESEARCH SUMMARY

In order to thrive in a changing environment all organisms need to ensure protein homeostasis (proteostasis). Proteostasis is ensured by the proteostasis system that monitors the folding status of the proteome and regulates cell physiology and gene expression to counteract any perturbations. An increased burden on the proteostasis system activates Heat shock factor 1 (Hsf1) to induce transcription of the heat shock response (HSR), a transiently induced transcriptional program including core proteostasis genes, importantly those encoding the Hsp70 class of molecular chaperones. The HSR assists cells in counteracting the harmful effects of protein folding stress and restoring proteostasis. The work presented in this thesis is based on experiments with the Saccharomyces cerevisiae (yeast) model with the overall goal of deciphering how Hsp70 detects and impacts on perturbations of cellular proteostasis and controls Hsf1 activity. In Study I we describe the fundamental mechanism by which Hsp70 maintains Hsf1 in its latent state by controlling its ability to bind DNA. We found that Hsf1 and unfolded proteins directly compete for binding to the Hsp70 substrate-binding domain. During heat shock the pool of unfolded proteins mainly consist of misfolded, newly synthesized proteins. Severe out-titration of Hsp70 by misfolded substrates resulted in unrestrained Hsf1 activity inducing a previously uncharacterized genetic hyper-stress program. More insight into regulation of Hsp70 availability was gained in Study II where the two splice isoforms of the Hsp70 nucleotide exchange factor Fes1 were characterized. We found that the cytosolic splice isoform Fes1S is crucial to release unfolded proteins from Hsp70 and that impaired release results in strong Hsf1 activation. In Study III we developed methodology to easily measure the rapid changes in Hsf1 activity upon proteostatic perturbations and to monitor protein turnover using the novel bioluminescent reporter NanoLuc optimized for yeast expression (yNluc). In Study IV we report that yNluc also functions as an in vivo reporter that detects severe perturbations of de novo protein folding by its failure to fold to an active conformation under such conditions. Finally, in Study V we investigated how organellar proteostasis impacts on the availability of cytosolic Hsp70. We found that a lowered mitochondrial proteostatic load as a result of high translation accuracy

i extended lifespan and improved cytosolic proteostasis capacity, evidenced by more rapid stress recovery and less sensitivity to toxic misfolded proteins. In contrast, lowered mitochondrial translation accuracy decreased lifespan and impaired management of cytosolic protein aggregates as well as elicited a general transcriptional stress response. Taken together, the findings presented in this thesis advance our understanding of how the regulatory mechanisms of the proteostasis system function. Furthermore, they provide novel methodology that will facilitate future studies to improve our understanding how cells integrate internal and external stress cues to control proteostasis.

ii POPULÄRVETENSKAPLIG SAMMANFATTNING

Alla levande organismer består av en eller flera celler som måste upprätthålla en inre balans, homeostas, för att må bra och växa. Ogynnsamma skiftningar i organismens omgivande miljö, som plötsliga temperaturhöjningar, påverkar cellerna negativt och kräver att de genomgår förändringar för att motverka eventuella skador samt anpassa sig till den nya miljön. En cell känner av plötsliga temperaturhöjningar genom att mängden felveckade proteiner ökar. Merparten av alla cellulära processer är beroende av proteiner vars funktion möjliggörs genom deras tredimensionella veckning. Därför är felveckade proteiner giftiga för cellen och måste hanteras för att återställa homeostasen. Chaperoner är proteiner vars uppgifter bland annat är att vecka, vecka om, stabilisera samt se till att felveckade proteiner bryts ned. En ökad mängd felveckade proteiner kräver fler chaperoner. Detta möjliggörs genom tillfällig aktivering av ett stressprogram som är bevarat från bakterieceller till människoceller. Stressprogrammet leder till att de felveckade proteinerna kan tas omhand så att cellens homeostas återställs. Denna avhandling fokuserar på hur den encelliga modellorganismen Saccharomyces cerevisiae (bagerijäst) kontrollerar sin homeostas genom reglering av stressprogrammet. Den beskriver hur en speciell chaperon, Hsp70, styr aktiviteten hos proteinet Hsf1 som reglerar stressprogrammet. Vilka de felveckade proteinerna är som rubbar homeostasen vid plötsliga temperaturändringar utreds också. Avhandlingen bidrar även med nyutvecklade metoder för att snabbt och enkelt kunna mäta aktiviteten hos de proteiner som aktiverar stressprogrammet samt mäta förändringar i cellers homeostas. Att förstå hur cellen säkerställer homeostas och aktiverar stressprogrammet är viktigt för förstå varför processen ibland går fel, vilket kan resultera i sjukdomar som cancer, Alzheimers och Parkinsons.

iii

iv LIST OF PUBLICATIONS

This thesis is based on the following studies:

I. Masser, A. E., Kang, W., Friedländer, M. R., & Andréasson, C. Cytoplasmic protein misfolding titrates nuclear Hsp70 to unleash active Hsf1. Manuscript

II. Gowda, N. K. C., Kaimal, J. M., Masser, A. E., Kang, W., Friedländer, M. R., & Andréasson, C. (2016). Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast. Molecular Biology of the Cell, 27(8), 1210–1219.

III. Masser, A. E., Kandasamy, G., Kaimal, J. M., & Andréasson, C. (2016). Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae. Yeast, 33(5), 191–200.

IV. Masser, A. E., & Andréasson, C. A co-translational folding reporter to monitor proteostasis. Manuscript

V. Suhm, T., Kaimal, J. M., Dawitz, H., Peselj, C., Masser, A. E., Hanzén, S., Ambrožič, M., Smialowska, A., Björck, M. L., Brzezinski, P., Nyström, T., Büttner, S., Andréasson, C., Ott, M. (2018). Mitochondrial translation efficiency controls cytoplasmic protein homeostasis. Cell Metabolism, 27(6), 1309–1322.e6.

v ABBREVIATIONS

CTA C-terminal activation domain CTD C-terminal domain DBD DNA-binding domain dHsf1 D. melanogaster Heat shock factor 1 ER endoplasmic reticulum ESR environmental stress response hHSF1 human Heat shock factor 11 HSE heat shock responsive element Hsf1 Heat shock factor 1 Hsp heat shock protein HSR heat shock response JDP J-domain protein KlHsf1 K. lactis Heat shock factor 1 LZ leucine-zipper domain mHSF1 mouse Heat shock factor 1 NBD nucleotide-binding domain NEF nucleotide exchange factor NES nuclear export sequence NLS nuclear localization sequence NTA N-terminal activation domain PKA protein kinase A PQC protein quality control RD release domain SBD substrate-binding domain STRE stress-responsive element TAD transcriptional-activation domain TF transcription factor TOR target of rapamycin UPR unfolded protein response UPS ubiquitin-proteasome system

vi CONTENTS

RESEARCH SUMMARY ...... i

POPULÄRVETENSKAPLIG SAMMANFATTNING ...... iii

LIST OF PUBLICATIONS ...... v

ABBREVIATIONS ...... vi

CONTENTS ...... vii

PREFACE...... ix

INTRODUCTION ...... 1 CELLULAR STRESS RESPONSES...... 1 YEAST AS A MODEL ORGANISM TO STUDY STRESS RESPONSES...... 2 PROTEIN FOLDING AND PROTEOSTASIS ...... 3 MOLECULAR CHAPERONES ...... 5 HSP70 ...... 5 HSP90 ...... 8 STRESS RESPONSIVE TRANSCRIPTION FACTORS ...... 10 HEAT SHOCK FACTOR 1 ...... 10 MSN2 AND MSN4 ...... 12 THE HEAT SHOCK RESPONSE ...... 14 SENSING STRESS ...... 14 THE TRANSCRIPTIONAL PROGRAM...... 14 THE ROLE OF HSF1 IN THE HEAT SHOCK RESPONSE...... 15 PROTEOME CHANGES UPON HEAT SHOCK ...... 17 HSF1 REGULATION ...... 19 E. COLI AS A MODEL TO UNDERSTAND HSF1 REGULATION ...... 19 SPATIAL REGULATION OF HSF1 ...... 20 REGULATION OF HSF1 BY PHOSPHORYLATION ...... 21 HSF1 AS A CELLULAR THERMOMETER ...... 22 REGULATION OF HSF1 BY CHAPERONE TITRATION...... 23 METHODOLOGY ...... 28 METHODS TO STUDY HSF1 ACTIVI TY ...... 28 METHODS TO STUDY HSP70 AVAILABILITY ...... 29

vii AIMS OF THIS THESIS ...... 31

SUMMARY OF INDIVIDUAL STUDIES...... 33 STUDY I ...... 33 STUDY II ...... 35 STUDY III ...... 36 STUDY IV ...... 37 STUDY V ...... 38

CONCLUDING REMARKS AND OUTLOOK ...... 41

ACKNOWLEDGEMENT...... 45

REFERENCES ...... 47

viii PREFACE

Coping with stress is a central aspect of an organism’s life. Whether looking at a unicellular or multicellular organism the requirement to adapt to changes in the environment is crucial for fitness and survival. This adaptation is achieved through inter- and intracellular communication involving some of the smallest building blocks of life; DNA, RNA and proteins. Understanding how cells manage stress to maintain an inner balance at the most detailed molecular level is the key to further reveal how failures can occur which may result in diseases such as cancer or neurodegeneration. This thesis aims at elucidating how Saccharomyces cerevisiae senses perturbations in protein homeostasis (proteostasis) and to reveal mechanistic details of its response, i.e. the mounting of a transcriptional stress response to counteract the harmful effects of protein misfolding. At the heart of this transcriptional stress response, the heat shock response (HSR), in organisms ranging from yeast to man lies the transcription factor Heat shock factor 1 (Hsf1). Accordingly, important questions regarding how proteostatic perturbations trigger Hsf1 activation and how its latency is maintained under non-stress conditions arise. In this thesis experimental data is presented that provide insight into how cells through protein-protein interactions and transcriptional reprogramming sense and respond to changes in the intracellular milieu. As part of the work, methodology has been developed that will ease further studies on Hsf1 and the system managing protein folding stress. The first part of this thesis puts cellular stress in a broader context by describing how it affects cells and what defence mechanisms cells activate to alleviate the stress. Next, the model organism S. cerevisiae is presented together with a discussion of why studies of protein folding stress in this organism are relevant for all eukaryotes. How protein folding stress affects proteostasis is then described. Molecular chaperones and their cofactors as an essential part of the proteostasis system are described in detail together with the transcription factors regulating them. Then, the HSR is introduced in detail. After that, previously proposed models for Hsf1 regulation are discussed to obtain an overview of earlier work on this important process. In addition, methodological approaches that have enabled the current understanding are explained.

ix The following two parts formulate the overall aim of this thesis as well as the individual aims for each of the five included studies. The studies are then introduced and discussed. Finally, a chapter with concluding remarks and perspectives reflects on important questions that remain to be answered to fully understand how cells regulate proteostasis.

x INTRODUCTION

CELLULAR STRESS RESPONSES All organisms experience stress when environmental fluctuations perturbs their homeostasis. An optimal non-stressful environment to promote cellular growth and division requires a stable temperature and a sufficient supply of nutrients. Stress can be triggered by both extrinsic and intrinsic events which results in damage to macromolecules forcing the cell to undergo changes in order to counteract the stress and restore homeostasis51. Cells respond to perturbed homeostasis by broad transcriptional responses that result in suitable physiological adaptations. Generally, a transcriptional response to stress is a rapid process where genes that support growth and division are repressed while genes involved in restoring energy metabolism, repressing apoptotic signals and counteracting macromolecular damage are induced48. Cells that already have encountered stress are more likely to survive further stresses. This phenomenon, referred to as acquired stress resistance has been observed for several different stresses in a wide variety of species including human cells36,95,157. Acquired stress resistance is the result of a common transcriptional response activated by different stressors. The broad transcriptional regulation program is therefore not only crucial to survive the primary stress but also to prepare for additional, more severe stresses17. The transcriptional and translational changes help cells to counteract the stress and restore homeostasis. Subjecting cells to a heat shock is a frequently employed experimental approach to perturb their homeostasis. The induced stress response, termed the heat shock response (HSR), has been widely studied which has helped researchers gain a fundamental understanding of the general mechanisms cells employ to cope with a changing environment as well as revealed the function of many genes83,126,142,187. In a broader context a rapid heat shock is an artificial stressor that can be viewed as an instant way to create stress in order to study factors normally involved in responses to a wide range of natural stresses. Although cells, whether unicellular or a part of a multicellular organism, experience fluctuations in temperature more typical stresses in nature include nutrient starvation and draught. However, stress adaptation to transient changes in the environment, even though they are more likely to occur at a slower rate, is crucial for all cells, hence making studies of the HSR important.

1 Heat shock induce a plethora of changes in the cell. It causes for instance a remodelling of the cytoskeleton leading to impairment of intracellular transport and mislocalization of organelles. Moreover, changes and damage to membrane structures negatively affects the endoplasmic reticulum (ER), Golgi and mitochondria148. Heat shock also affects cell cycle progression; cells are prevented from proceeding from G1 to S phase thereby inhibiting genome replication87,151,185. Furthermore, the amount of proteins losing or failing to reach a functional folded state dramatically increase in the cell. All these physiological changes and the mechanisms behind them can be studied by heat shocking cells thus providing important information about stress responses and physiological stress adaption.

Figure 1. Physiological changes in the cell upon heat shock. Heat shock gives rise to perturbations of intracellular transport and membrane integrity. Cell cycle progression is inhibited and the cell will experience an increased amount of misfolded proteins.

YEAST AS A MODEL ORGANISM TO STUDY STRESS RESPONSES The budding yeast Saccharomyces cerevisiae (yeast) is a member of the Ascomycota phylum of the Fungi kingdom and a widely used model organism. In the wild, yeast has been frequently isolated from oak bark and ripening fruit but also from other plants and the soil surrounding them along with insects associated with the plants. Its ability to ferment sugars into ethanol has made it popular among humans and it is consequently found in human surroundings as well as in the human gut. One of the main benefits of yeast as a model organism is that it easily can be cultured in a laboratory environment. Importantly, together with the fact that yeast has many similarities with multicellular eukaryotes when it comes to gene expression, intracellular organization and differentiation have made it a powerful tool to study cellular stress responses and the involved transcriptional mechanisms. Although not all the molecular mechanisms of human cells are represented in yeast cells, its study has over and over again revealed the basic molecular

2 principles of eukaryotic cells of relevance for human physiology, health and treatment of disease. In general, many processes are performed in a less complex way than in human cells and involve fewer components but importantly the basic mechanism have often remained conserved throughout evolution. This makes yeast a good starting organism to study novel biological processes103. The yeast genome was the first eukaryotic genome to be sequenced and many of the ~6000 genes have been well characterized with the help of deletion libraries, overexpression mutants and genes tagged with reporter genes54,57,79,173. The extensive characterization of many yeast genes makes it a good reference to discover orthologues in other organisms. For example, identification of novel human genes involved in chromosome integrity were isolated in a yeast screen by complementation of known essential yeast genes involved in the process70. Proteins not present in yeast can be studied in it using ectopic expression. For instance, all possible point mutations in the human tumour suppressor gene p53 were screened for function and activity in yeast despite that orthologues do not exist93. The possibility to study the function of endogenous yeast genes as well as other genes through the use of plasmids and chromosomal modifications by homologous recombination, makes yeast a powerful tool for genetic experiments and a valuable model for studies of diverse biological processes including how cells counteract the harmful effects of stress.

PROTEIN FOLDING AND PROTEOSTASIS Proteins are synthesized as linear structures consisting of one amino acid after another that need to adopt a correct three-dimensional state in order to function properly. All the information required for a protein to achieve its correct three-dimensional fold is encoded by its primary amino acid sequence11. A typical soluble protein consists of a hydrophobic core shielded by a hydrophilic shell which is the most favourable conformation in the hydrophilic cellular milieu. Proper folding often requires a whole domain or even the entire protein to be synthesized which results in stretches of exposed hydrophobicity during translation. In a crowded cellular environment unprotected hydrophobicity may cause unproductive interactions between proteins resulting in failure to fold and the start of protein aggregation186. For this reason, many proteins require help by other proteins in order to attain a correctly folded state. This is managed by molecular chaperones, a group of highly conserved proteins whose role in the cell is to assist in the formation of non-covalent polypeptide interactions without being a part of the final protein structure72. A correctly folded protein is not a rigid structure but a dynamic entity whose function often relies on its ability to adopt different conformations. One example is the microfilament system where monomeric actin through conformational

3 changes driven by ATP hydrolysis can form dynamic filamentous structures spanning entire cells. Also, many receptors e.g. steroid receptors keep an open conformation to facilitate ligand binding. Ligand binding drives conformational changes and results in receptor activation19,141. The flexibility of proteins causes them to sometime need assistance from other proteins to maintain a productive, functional folded state thereby preventing unfolding and aggregation. Increased protein misfolding affects protein homeostasis (proteostasis). Proteostasis refers to the cellular process of controlling the concentration, conformation, interactions and localization of proteins through mainly transcriptional and translational changes13. Maintaining proteostasis is crucial for the cell and any proteostatic perturbations have to be taken care of. Proteostasis is maintained by the proteostasis system which assist in protein synthesis, folding, trafficking, disaggregation and degradation of proteins13. The proteostasis system is a complex network involving molecular chaperones and their co-factors, the ubiquitin-proteasome system (UPS) and autophagy components71. Heat stress puts extra strain on the proteostasis system as it causes proteins to enter unfolded or misfolded states that promote aggregation. Proteins that are not correctly folded cannot perform their normal cellular functions resulting in collapse of downstream cellular processes which in turn results in further stress. Cells employ several mechanisms to counteract a proteostasis imbalance following a stress14,71. One is to increase the amount of proteins involved in the proteostasis system which enables the cell to take care of unfolded, misfolded and aggregated proteins by either refolding, degrading or sequestering them so that proteostasis can be restored.

Figure 2. De novo folding of a protein. The protein is translated as a linear chain of amino acids that is protected by chaperone binding to hydrophobic sequences. Chaperones aid in the folding of the protein until it has achieved its correct three- dimensional fold.

4 MOLECULAR CHAPERONES Molecular chaperones are necessary for protein biogenesis by taking part in their de novo folding but also to aid in processes such as protein translocation across membranes, aggregation prevention and refolding or degradation of proteins14. Not all molecular chaperones are structurally related to each other and therefore they are traditionally divided into classes based on their molecular weight. In yeast the major chaperone families are Heat shock protein 70 (Hsp70), Hsp90 and Hsp100, and chaperonins. They recognize and bind exposed hydrophobic residues on client proteins. With the assist of cochaperones and ATP to drive conformational changes of the client protein they help it achieve its final fold. The cochaperones include nucleotide exchange factors (NEFs), J-domain proteins (JDPs) and small heat shock proteins (Hsps)71,98. NEFs aid in the release of ADP from chaperones while JDPs facilitate substrate delivery and ATP hydrolysis. Small Hsps function in an ATP-independent manner by holding on to unfolded proteins thereby preventing aggregation. The following parts describe in more detail the Hsp70 and Hsp90 chaperone families that are conserved from bacteria to man and play major roles in cellular stress sensing, recovery and adaptation. In addition, some of the Hsp70 cofactors are described.

HSP70 Hsp70 is a class of molecular chaperones performing numerous tasks to assure proteostasis thereby playing an important role in stress survival and development of thermotolerance120. Hsp70 acts on proteins throughout their lifetime by transient binding to exposed segments of 5 hydrophobic amino acids152. This protects the client protein from forming unproductive hydrophobic interactions with other proteins in the crowded cellular environment but can also prevent its spontaneous folding. After Hsp70 interaction there are several paths the protein can embark on including spontaneous folding on its own, transferral to Hsp90 for further assisted folding or being targeted for degradation125. The structure of Hsp70 consists of a highly conserved N-terminal nucleotide-binding domain (NBD), a substrate-binding domain (SBD) and a variable C-terminus119. The affinity for substrates is low when Hsp70 is bound to ATP. ATP hydrolysis stimulated by substrate interaction and JDPs enhances substrate affinity causing a tighter binding. Simultaneously, the C-terminal domain undergoes a conformational change causing it to act as a lid over the substrate-binding pocket. ADP release, facilitated by NEFs, and subsequent spontaneous re-binding of ATP opens the substrate-binding pocket again resulting in lowered substrate affinity and completes the Hsp70 cycle. In addition, eukaryotic cytosolic Hsp70s contain an EEVD motif

5 which enables interactions with TPR-domains that are found in many cochaperones. In yeast, there are in total seven cytosolic Hsp70 members. Four of them, Ssa2, Ssb1, Ssb2 and Ssz1 are constitutively expressed and play crucial roles in the proteostasis network at all times. Proteotoxic stress puts extra pressure on the Hsp70 system as the fraction of misfolded protein increases as well as the amount of proteins needing chaperone maintenance to remain soluble. In response to stress, Ssa1, Ssa3 and Ssa4 are induced to provide further assistance in restoring proteostasis42,73.

J-DOMAIN PROTEINS J-domain proteins (JDPs) are a class of proteins around 40 kDa in size that bind to unfolded proteins thereby preventing their aggregation and facilitating contact with Hsp70. Apart from the highly conserved J-domain, JDPs have diversified through evolution into a structurally varied group thereby providing an extra layer of regulation to Hsp7090. JDPs bind via their HPD motif to the linker between the NBD and SBD of Hsp70 when it is in its open, ATP-bound conformation99. Binding results in substrate transfer from JDPs to Hsp70 and triggers ATP hydrolysis thereby increasing Hsp70- substrate affinity. Some JDPs are constitutively expressed whereas others, such as yeast Ydj1 and Sis1 are induced upon stress.

NUCLEOTIDE EXCHANGE FACTORS Nucleotide exchange factors (NEFs) are co-factors that assist in the release of ADP from another protein, e.g. chaperones, thereby facilitating binding of ATP21. Hydrolysis of ATP to ADP is used by chaperones as a source of energy to drive conformational changes. The conformational changes are of great importance to fold nascent chains and to refold unfolded or aggregated proteins. Fes1, HspBP1 in mammals, Sse1, Sse2 (Sse1/2) and Snl1 are the four cytosolic NEFs of the Hsp70 system in yeast. Fes1 was initially discovered as a cytosolic homologue of Sil1, a NEF for the endoplasmic reticulum (ER) Hsp70 Kar288. The structure of Fes1 consists of four α-helical armadillo repeats and a N-terminal flexible release domain (RD)63,166. The armadillo domain enables contact between Fes1 and the Hsp70 NBD to facilitate ADP release while a second interaction between the RD on Fes1 and the SBDβ of Hsp70 aid in the release of persistent Hsp70 substrates63,166. Substrate release is followed by substrate degradation via the ubiquitin-proteasome system (UPS)61,62. Fes1 is important for the Hsp70 Ssa1-4 folding cycle and deletion of the FES1 gene results in temperature-sensitive growth at elevated temperatures, due to accumulation of misfolded proteins and failure to release them from Hsp701,62,88. Both deletion of the RD and two point mutations in the armadillo domain that also abolish Hsp70 NBD binding mimic the knock-out phenotype showing that both the RD and armadillo

6 domain interactions are essential for Fes1 function62,63,166. The accumulation of misfolded proteins causes fes1Δ cells to exhibit a constitutively induced stress response thus making it a potential model to study cellular responses to an extended stress. The Hsp110 family of chaperones consists of Sse1 and Sse2 (Sse1/2) in yeast which are the most abundant cytosolic NEFs52. Sse1/2 were isolated in a screen for calmodulin-binding proteins based on the notion that the Hsp70 family of proteins contained such a domain127,178. Sse1/2 are 77.4 and 77.6 kDa respectively and share 97% amino acid sequence similarity with each other as well as 70% amino acid sequence similarity to Ssa1127. Similarly to the Hsp70 family, Sse1/2 contain an N-terminal NBD with ATP-ase activity133,164. However, in contrast to Hsp70, Sse1/2 contain a negatively charged insertion between SBDβ and SBDα characteristic to the Hsp110 family of chaperones112,133. The Sse1 structure is stabilized by nucleotide binding where ATP is preferred over ADP. Moreover, nucleotide binding is required for Sse1 to interact with Hsp709,10. ATP binding to Hsp70 results in disassociation of the complex10. Sse1 is more abundant than Sse2 and deletion of SSE1 results in slow growing, temperature sensitive cells whereas deletion of both isoforms is lethal127,146. The Sse1 NBD interacts with both Ssa and Ssb to provide release of ADP10,146,164. Sse1/2 is required for many cellular processes involving Hsp70 such as delivery of substrates to the proteasome and Hsp104 mediated disaggregation of protein aggregates89,91. The fourth NEF in yeast is represented by the 18.3 kDa protein Snl1 belonging to the BAG-domain family of proteins172. Snl1 is the least abundant NEF in yeast and deletion of SNL1 does not result in any clear phenotype which has made it difficult to assign any function to the protein1,52. Snl1 contains a transmembrane domain that tethers it to the ER with a large part of the protein facing the cytosol172. The BAG-domain is required for its ATPase activity and for mediating contact with the Hsp70s Ssa and Ssb172. Also, a lysine-rich region within the domain is able to contact the 60S ribosomal subunit for the formation of a Snl1-Ssb-60S complex188.

Figure 3. Folding of a protein via Hsp70 and Hsp90. Hsp70-ATP has low substrate affinity. ATP to ADP hydrolysis stimulated by the substrate and J-domain proteins results in tighter binding and promotes conformational changes of the substrate. Nucleotide exchange factors aid in release of ADP followed by spontaneous ATP rebinding which completes the cycle. The substrate can go through several rounds of Hsp70-mediated folding until it is transferred to Hsp90 by cochaperones interacting simultaneously with Hsp70 and Hsp90. ATP binding to the Hsp90 dimer causes it to adopt a closed conformation trapping the substrate. Subsequent ADP hydrolysis by cochaperones results in an open, V-shaped Hsp90 conformation where the substrate can be released. ADP release and ATP rebinding concludes the cycle. After Hsp90 cycling the substrate can proceed to fold and mature on its own. Adapted from Mayer & Bukau (2005) and Taipale et al. (2010).

HSP90 Hsp90 works downstream of Hsp70 in the folding of proteins to their native conformation. The cochaperone Hop (Sti1 in yeast) bridges the two chaperones by interactions via its TPR-domains hence facilitating substrate transfer150. In contrast to Hsp70, Hsp90 substrate recognition is not dependent on short hydrophobic amino acid stretches. Instead, Hsp90 associates with more spread out hydrophobic and negatively charged residues on the protein, thereby functioning as the logical partner for a protein that already has been partially folded by Hsp70124. After going

8 through multiple rounds of Hsp90 interactions a protein can normally fold and mature by itself125. The Hsp90 folding cycle is driven by ATP and the interaction of cochaperones that also aid in substrate delivery. Hsp90 consists of a C-terminal domain (CTD) involved in the formation of Hsp90 dimers giving rise to their V-shape 137. The CTD also harbours a TPR domain important for cochaperone interactions. The N-termini of the dimer are able to form transient contacts upon ATP binding which closes the V-shape thereby trapping substrates and triggering ATP hydrolysis143. The N-terminal domain contains a NBD followed by a flexible linker that connects it to the middle domain. The middle domain is the binding site for substrates and certain cochaperones and is important for ATP hydrolysis. Apart from taking over substrates in need of folding from Hsp70, Hsp90 also aids in stabilizing native proteins158. Steroid receptors are one of the most studied examples where Hsp90 binding provides needed stability to the latent aporeceptor complex prior to hormone binding182. Hormone binding to the receptor stabilizes it resulting in Hsp90 disassociation and translocation of the active receptor to the nucleus where it induces transcription19,46,141. Hsp90 is also important for stabilization of many kinases in order for them to exert their normal function145. As a consequence, destabilizing kinase mutations found in many cancers can be buffered by Hsp90 thereby preventing them from degradation making Hsp90 a potential target in anti- cancer therapy196.

9 STRESS RESPONSIVE TRANSCRIPTION FACTORS Heat stress triggers broad transcriptional changes in cells aimed at restoring proteostasis. In eukaryotes, the process of transcription is under control of a great number of regulatory proteins such as RNA polymerase, chromatin- remodelling complexes, histone acetylases and transcription factors (TFs). TFs are DNA-associated proteins that control the rate of transcription by facilitating the recruitment of RNA polymerase. Many TFs bind directly to DNA-sequences, so called response elements, in the promoter region of genes whereas others do not bind to DNA directly but instead are a part of the large transcription preinitiation complex. Regulation of TF activity can occur on several levels and is an important step in transcriptional regulation. Transcription and later translation of the TF itself is one way of controlling the amount of available TF protein. Since transcription takes place in the nucleus, regulation of nuclear localization or sequestering of the TF is another step to control its activity. Finally, additional steps such as ligand binding, oligomerization or post-translational modifications may be needed for TF activation. The heat shock response (HSR) is an ancient gene-regulatory program that is induced when cells are subjected to an elevated temperature, such as a heat shock. In yeast, it is mainly governed by the three TFs Heat shock factor 1 (Hsf1), Msn2 and Msn4 (Msn2/4)187. They are DNA binding TFs with a low basal activity under non-stressful conditions. Upon heat shock their increased activity results in rapid induction of a broad range of genes required by the cell to restore proteostasis.

HEAT SHOCK FACTOR 1 The heat shock factor family is a conserved family of transcription factors found in all eukaryotes, comprised of only one Heat shock factor (Hsf) in invertebrates but multiple factors in mammals and birds4,113. Traditionally, Hsf has been studied using heat as the main stressor, hence the name heat shock factor. However, Hsf can be activated by a variety of stresses including oxidative-, osmotic- and ethanol stress as well as nitrogen starvation and toxic analogues of amino acids. Hsfs bind as a trimer to heat shock responsive elements (HSE), a repeated nGAAn DNA sequence, to induce transcription175,176,198. HSF1 in yeast is an essential, single copy gene that encodes a 93 kDa transcription factor with several functional domains197. A long N-terminal activation domain (NTA), 170 amino acids in yeast, is a unique Hsf1 feature in unicellular eukaryotes that is not present in e.g. Drosophila melanogaster Hsf1 (dHsf1) or mammals. Nuclear magnetic resonance spectroscopy has shown that the domain is unstructured, which presumably is why it has not

10 yet been crystalized27. The NTA is required for transient induction of Hsf1 activity131,174. C-terminally of the N-terminal activation domain is a DNA- binding domain (DBD) of the winged helix-turn-helix family197. This domain is the most conserved in the heat shock factor family across all eukaryotes and the only domain that has been structurally determined130. The importance of the DBD can be exemplified with the fact that Kluyveromyces lactis Hsf1 (KlHsf1), a protein carrying only 18% amino acid conservation with Hsf1 is able to complement its function in yeast81. Presumably, this is because the DBD carries a 73% amino acid conservation and an even higher degree of conservation in terms of amino acid similarity. Via a flexible linker the DBD is connected to an α-helical leucine-zipper domain (LZ), which contains three leucine zippers, also known as HR-A and HR-B, required for trimerization139,175. Trimerization is crucial for Hsf1 activity and in yeast a pool of Hsf1 is trimerized at all times175. When expressed in yeast, human HSF2 is constitutively trimerized and can complement Hsf1 function, a feature that human HSF1 (hHSF1) does not posess113. However, hHSF1 carrying four point mutations, which lead to constitutive trimerization can complement Hsf1 function113. C-terminal of the trimerization domain is a regulatory domain containing a sequence conserved between K. lactis and yeast, CE2, required for the repression of basal Hsf1 activity77,81,101. The C- terminal activation domain (CTA) is necessary for sustained Hsf1 activation and followed by a fourth LZ. Deletion of the CTA results in temperature sensitive growth at 37°C26,174.

Figure 4. Heat shock factor 1 (Hsf1) and its domains. Hsf1 contains an N-terminal activation domain (NTA) followed by a DNA binding domain (DBD), Leucine zipper 1-3 (LZ1-3), a regulatory domain containing CE2, a C-terminal activation domain (CTA) and finally a fourth leucine zipper (LZ4). Hsf1 is subjected to several post-translational modifications. The most studied one is phosphorylation but also acetylation and sumoylation events have been reported in multicellular eukaryotes4. Hsf1 contains 153 putative phosphorylation sites in the form of serine and threonine residues. Three high throughput screens identified 24 phosphorylated residues6,78,180. Another screen using various growth conditions and stressors identified 73 phosphorylation sites, 19 found previously but also 54 novel sites spread across all domains205. This means that phosphorylations have been detected on approximately half of all serine and threonine residues. The amount of phosphorylation increases upon heat shock or other stresses and has

11 therefore been seen as an important step in the regulation of Hsf1 activity which will be discussed later on in the thesis177.

MSN2 AND MSN4 MSN2 and MSN4 are yeast-specific genes encoding two TFs, Msn2 and Msn4 (Msn2/4). The genes are non-essential but deletion of both genes results in decreased stress resistance44. Msn2/4 were first discovered as multicopy suppressors of a temperature-sensitive SNF1 allele, an essential serine/threonine kinase related to mammalian AMPK and required for growth during low glucose levels45. Msn2 is constitutively expressed and more abundant than Msn4 whose expression is activated by stress. Msn2/4 are the main inducers of the environmental stress response (ESR), a broad general transcriptional program that overlaps with the HSR49,76. The two proteins have overlapping functions but, depending on the stressor, are not always redundant5,17. Msn2 and its paralog Msn4 are similar in size, 78 kDa and 70 kDa respectively, and share 41% amino acid sequence similarity. The proteins are largely unstructured so no 3D structure have been solved. The N-terminal domain of Msn2 harbours a transcriptional-activation domain (TAD) essential for function104,154. The most N-terminal part of the TAD contains a region predicted to form a secondary structure154. It is followed by a nuclear export sequence (NES) that is highly conserved among Msn2 orthologues and contains a phosphorylation site59,140. Two conserved domains follows, HD3 and HD2 of unknown function where HD2 is predicted to form an α-helical structure140,154. Towards the C-terminus lies a nuclear localization sequence (NLS) with multiple phosphorylation sites. The very C-terminal part of the proteins harbours a DBD consisting of two zinc finger motifs that have 100% amino acid similarity between Msn2/4. The DBD recognizes stress-responsive elements (STRE), a CCCCT sequence found upstream of a broad variety of genes involved in the HSR118,156.

Figure 5. Msn2 domains. Msn2 consists of an N-terminal trans-activation domain followed by a region containing an NES, two domains conserved among Msn2 orthologues denoted HD3 and HD2, an NLS and lastly a C-terminal DNA-binding domain. Msn2/4 are regulated by protein kinase A (PKA) and target of rapamycin (TOR) signalling events. Under non-stressful conditions Msn2/4 are kept in

12 a repressed state by mechanisms regulating their localization and phosphorylation. They are localized to the cytosol due to inhibition of their NLS by phosphorylation facilitated by Tpk1-3, the catalytic subunits of PKA59,60. The small pool of nuclear Msn2/4 capable of activating transcription is efficiently exported through its NES by the nuclear export receptor Msn5106. TOR regulates Msn2/4 through the cytosolic 14-3-3 protein Bmh2 which sequesters Msn2/4, thus preventing them from entering the nucleus16. These regulatory steps result in repression of Msn2/4 activity and thus in low induction of STRE genes when glucose is available. Upon stressful conditions or when cells change to a non-preferred carbon source (diauxic shift), PKA levels drop due to reduced glucose uptake resulting in less phosphorylation and thus activation of the NLS in Msn2/4. Yak1, a kinase kept inactive by PKA at non-stressful conditions, gets activated by lower PKA levels. Yak1 phosphorylates Msn2/4 which inhibits its nuclear export by Msn5 causing Msn2/4 to accumulate in the nucleus where it can induce transcription of STRE genes106,107. However, nuclear accumulation of Msn2/4 by deletion of MSN5 does not result in increased transcription of STRE genes39. Instead, constitutive activation of Msn2 can be achieved by six serine to alanine mutations spanning the NES, NLS and DBD resulting in transcriptional activation resembling diauxic shift140. Another layer of regulation is maintained through the degradation of Msn2/4 in the nucleus by the 26S proteasome. Degradation is dependent on DNA binding, leading to a shorter half-life for the proteins upon stress105. In short, tight control of Msn2/4 activity is ensured through multiple regulatory mechanisms controlling their localization, phosphorylation state and degradation rate.

13 THE HEAT SHOCK RESPONSE The heat shock response (HSR) is a universal transcriptional response present in most living organisms. It is activated when an organism is exposed to changes in temperatures that may only vary by a few degrees but can have a strong negative impact on the proteome. Sensing and adapting to an elevated temperature is a multi-step process involving several cellular machineries and signalling pathways. Therefore, understanding how cells achieve this has been an aim for researchers during several decades. It requires first identification of the stressor that triggers the response and second the early responders to the stress. Then the sequential steps that cells employ in order to restore proteostasis and prepare for further insults have to be revealed.

SENSING STRESS Independent of the stressor used to elicit a stress response the outcome will be an increase in heat shock proteins (Hsps) hence implicating unfolded proteins as the factor triggering the HSR. Specifically, induction of co- translational protein misfolding by toxic amino acid analogues or drugs enhanced Hsp synthesis and further supported the idea37,64,94. Direct evidence was obtained when native or denatured proteins where injected into Xenopus oocytes together with a reporter for HSP70 activity, where only the denatured proteins activated the reporter8. Some proteins are more prone to unfolding and misfolding than others190. In general, rapidly growing cells with high translational rates are more sensitive to stress whereas stationary phase and slow growing cells show increased stress tolerance41,115,194. Blocking translation prior to stress results in formation of fewer protein aggregates suggesting that newly synthesized proteins are most aggregation prone207. Newly synthesized proteins are also the main pool of proteins targeted for degradation upon stress indicating that they are not properly folded and functional121. In yeast, it was estimated that the fitness cost of ectopic expression of a misfolded model protein representing less than 0.1% of the proteome led to a decrease in growth by 3.2% and was enough to induce a stress response50. Thus, unfolded proteins are activators of the HSR, a multi-step process aiming at detoxifying the unfolded proteins to restore proteostasis.

THE TRANSCRIPTIONAL PROGRAM Heat-shock treatment of cells has been instrumental for the identification of the HSR and the factors involved in its regulation. Ferruccio Ritossa was the first scientist to report on changes in gene expression following heat shock in D. busckii. In chromosome puffs, i.e. regions with high ongoing transcription

14 on polytene chromosomes in the salivary glands, he saw a different puffing pattern when the temperature was increased149. Since then, the HSR has been studied in all kingdoms of life and the level of conservation in the transcriptional response between both species and kingdoms has been found to be remarkably high18,33,109,134. The HSR in yeast is a rapid broad transcriptional response that depending on severity and metabolic state of the cells may affect transcription of up to half of the genome. Induction of the HSR includes both induction and repression of genes spanning several functional groups. Heat shock causes uncoupling of oxidative phosphorylation resulting in a sudden drop in ATP levels and changes in membrane fluidity lowers the intercellular pH thereby affecting the proton gradient. Lowered ATP and pH levels cause the intracellular milieu to undergo rapid changes and making cellular metabolism the largest functional group regulated by the HSR47. Primarily, genes regulating protein synthesis, ribosomal proteins and RNA metabolism are repressed resulting in decreased cellular growth. Conversely, genes involved in fatty acid and carbohydrate metabolism are induced. In yeast, glycogen and the disaccharide trehalose have dual roles upon heat shock as they both stabilize proteins and function as an energy reserve136,168. The second largest group of genes is involved in controlling proteostasis; the increased fraction of unfolded and misfolded proteins either has to be refolded, degraded or sequestered, hence cells require more chaperones, ubiquitin-proteasome system proteins and autophagy components to be produced. Other functional groups include genes encoding RNA- and DNA- modifying enzymes that participate in DNA repair and management of reactive oxygen species, processes that are crucial to maintain genome integrity after stress. Regulatory proteins such as transcription factors and kinases that in turn change gene expression play a pivotal role in communicating the stress. A final group consists of genes encoding proteins involved in stabilizing cellular structures, i.e. cytoskeletal genes and genes that maintain membrane integrity48,69. Together, all these changes prompted by the HSR allow the cell to adapt to an elevated temperature by restoring proteostasis.

THE ROLE OF HSF1 IN THE HEAT SHOCK RESPONSE Hsf1 is often described as the “master regulator of the HSR”. While this may be true, the parallel transcriptional regulation of many of the heat-shock responsive promoters by several TFs complicates the mechanistic interpretation of experiments involving a heat shock. This can be illustrated by a study that aimed to elucidate the respective roles of the N- and C- terminal activation domains of Hsf1 upon heat shock40. When expressing Hsf1 mutants, significant differences were only seen in the absence of Msn2/4 supporting the notion that impairment of Hsf1 can be compensated

15 for by Msn2/4 and that they have overlapping roles in the transcriptional response to stress22,40. Detection of Hsf1 binding to promoters on individual genes revealed an increased heat shock responsive element (HSE) occupancy upon heat shock compared to low stress conditions55. Moreover, on a whole- genome level increased Hsf1 binding was detected upstream of 140 genes68. However, a chemical genetics approach to tether Hsf1 away from the nucleus showed that the HSR remained largely unperturbed in the absence of nuclear Hsf1171. Instead, Msn2/4 were found to regulate a majority of all induced genes upon heat shock171. In mice, where mHSF1 is not essential, its deletion resulted in a HSR that was 87% similar to WT117. The similarity in responses was not due to compensatory effects by the ubiquitously expressed mHSF2117. Consequently, even though Hsf1 is essential in yeast at all temperatures and Msn2/4 is not, the role of Hsf1 in the HSR is not as prominent as previously stipulated. Hsf1 might have an important role in facilitating rapid translation of transcripts. Not all the mRNAs of all the genes transcribed during stress are immediately translated. Also, transcripts produced pre-heat shock are sequestrated into P-bodies and degraded to facilitate the rapid induction of stress-responsive mRNAs135. Still, a bulk of newly produced mRNA accumulate in the nucleus upon heat shock whereas other, such as the molecular chaperones of the Hsp70 family Ssa1 and Ssa4 are successfully exported and translated102,153. Selective mRNA export upon glucose starvation has been shown to depend upon certain regulatory sequences in the promoter regions208. Specifically, genes under Hsf1 regulation were exported and selected for translation whereas Msn2/4 regulated genes, although they were exported, accumulated in stress granules208. Stress granules consist of mRNAs and translation initiation factors allowing for translation once the stress is relieved and thereby providing another layer of regulation at the transcriptional level24. Bypass of mRNA quality control and no requirement for RNA shuttling adaptor proteins have been proposed as the underlying mechanism behind selective nuclear export of HSE- containing genes upon heat shock203. Instead, these mRNAs were co- transcriptionally loaded with a nuclear export protein and directly exported203. Thus, despite the minor role of Hsf1 in the HSR it may serve as an important factor in terms of determining which mRNAs leave the nucleus and are translated.

Figure 6. Adapting to stress. The stress sensor and the stressor, represented here by a chaperone responding to unfolded proteins, results in transcription factor activation and transcription. Transcribed genes are then translated into proteins to act on the unfolded proteins. The cycle will continue until the cell has recovered from the stress or adapted to the new conditions.

PROTEOME CHANGES UPON HEAT SHOCK Even though heat shock affects transcription of a plethora of genes, proteins are the main functional entities of the cell. Consequently, it is not until the mRNA is translated and the produced protein has acquired its correct fold that the cell is able to fully adapt to new conditions. The translated proteins will help the cell gain stress resistance to further insults even if it is not the same type of stress, referred to as cross resistance181. Stress induction by amino acid analogues is the only stress found so far that does not result in cross resistance108. A large portion of the proteins produced during such stress will have the structurally perturbing amino acid analogue incorporated and therefore will not be able to adopt their native, functional conformation needed for stress recovery and adaptation. This shows the importance of changing the proteome in order to combat proteostasis imbalances. In contrast to changes in mRNA levels that occur just minutes after heat shock, effects on the proteome take longer time. Proteotoxic stress as a result of heat shock does not interfere with translation initiation, i.e. eIF2 activity111. Instead, translation elongation time is affected, as low Hsp70 availability results in translational pausing when the nascent chain exits the ribosome tunnel111,163. A proteome wide study investigating changes during 4 hours after a mild temperature upshift found that significant changes in the proteome were few after the first 30 min82. Still, 15% of the detected proteome was up- or down regulated at some point during the stress compared to before it82. Of the differently expressed proteins, approximately 80% were upregulated and included proteins involved in refolding,

17 carbohydrate metabolism and heat responsiveness. The first functional group of induced proteins represents many chaperones, notably small Hsps and the disaggregase Hsp104 were among the top hits82. Tps1-3 and Tsl1, enzymes involved in trehalose synthesis were a part of the second functional group. The 20% of downregulated proteins included proteins of functional groups related to cell growth and protein synthesis82. One major transcriptionally downregulated functional group upon stress were genes involved in ribosome biogenesis, a group also downregulated on the proteome level48,82. However, ribosomes are stable entities with long half-lives so it is unclear if the downregulation will give long-term effects on total ribosome levels. Finally, it is important to note that many heat-shock induced genes and proteins are not required for survival during the acute stress but instead contribute to acquired stress resistance so that the cell may survive further insults.

18 HSF1 REGULATION The function of Heat shock factor (HSF) homologues has diversified through eukaryotic evolution to drive transcription related to diverse processes. Many of these processes, for instance those related to the heat shock response and development are transient responses that needs to be coordinated and executed at a given time. Therefore, HSFs have to be kept under tight regulation. In birds and mammals expressing multiple HSFs, HSF1 is important upon stressful conditions whereas other HSFs manage basal induction of chaperones189. Moreover, mouse (mHSF1) plays a role in zygote development29. The sole D. melanogaster Hsf1 (dHsf1) is essential for oogenesis, early larval development and stress responsiveness84. Hsf1 is essential in yeast at all temperatures as it drives basal expression of HSP70 and HSP90 genes171,197. Given the multiple roles of Hsf1 homologues their regulation might differ between species. Consequently, several models for Hsf1 regulation have been proposed that will be discussed below.

E. COLI AS A MODEL TO UNDERSTAND HSF1 REGULATION Even though Hsf1 is not present in bacteria the study of the HSR in E. coli has provided the paradigm for understanding its regulation. In bacteria stress-induced transcription is regulated by two sigma factors, σ32 and σE, which respond to intracellular stress and outer membrane stress respectively. σE is bound by its anti-σ factor RseA, a membrane protein, at low temperatures. Upon heat shock σE is activated by a series of signalling events resulting in cleavage and degradation of RseA as a result of cell wall remodeling123. σE activated genes includes rpoH which codes for σ32 43. σ32 is repressed by three different mechanisms during physiological temperatures. On the translational level, secondary structure formation of the σ32 mRNA results in blockage of its ribosome binding site thus inhibiting translation. On the post-translational level σ32 is kept inactive by binding to the two main chaperone systems in bacteria; the DnaK machinery and GroEL/ES that have a low protein-folding burden under low stress. Lastly, σ32 is rapidly degraded by proteases, notably FtsH that degrades σ32 in a DnaK-dependent manner183. During heat shock the mRNA secondary structure is eliminated resulting in higher levels of σ32 protein. The chaperones are occupied with increasing amounts of unfolded and misfolded proteins resulting in a decreasing capacity to bind and inhibit σ32. As more unfolded and misfolded proteins are targeted for degradation the proteases become saturated, leading to an increased half-life of σ32 from 1 to 10 min161. Together, this results in rapid, transient σ32 activation and induction of the HSR. Importantly, the chaperone titration of a factor that controls transcription has provided the paradigm for understanding how Hsf1 is regulated in eukaryotes.

Figure 7. Model for chaperone titration of σ32 in E. coli. Under non-stressful conditions σ32 latency is maintained by DnaK binding. Upon proteotoxic stress the amount of unfolded proteins increase thereby competing for DnaK binding with σ32. Free σ32 can bind DNA and induce the heat shock response.

SPATIAL REGULATION OF HSF1 In contrast to bacterial σ32, eukaryotic HSFs are stable proteins. Yeast Hsf1 displays a half-life of over 10 hours, thus requiring other ways of regulation than translation of the transcription factor (TF) itself28. The control of activity through shuttling between the nucleus and the cytosol is an important mechanism in Hsf1 regulation in higher eukaryotes4. Human HSF1 (hHSF1) is kept inactive by sequestration of the monomers away from the nucleus within the cytosol via unknown mechanisms155. Upon stress, hHSF1 is transferred to the nucleus where it trimerizes and activates transcription155. In contrast, a single dHsf1 is found in the nucleus even in the absence of stress but DNA binding and thus transcription does not occur until the fly is subjected to stress195. Yeast Hsf1 is nuclear, trimerized and a fraction of its expressed population is DNA bound both in the absence and presence of stress80,138,175,176. Thus, cytosolic sequestration of Hsf1 provides only one layer of regulation in controlling Hsf1 latency.

20 REGULATION OF HSF1 BY PHOSPHORYLATION In contrast to bacteria post-translational modifications are common in eukaryotes providing another layer of Hsf1 regulation. Increased phosphorylation of Hsf1 is associated with its activation in proportion to the severity of the stress. However, it is not essential for transcriptional activation in vitro177. Phosphorylation increases the acidity of the protein due to the negative charge of the added phosphoryl group. For transcription factors and upstream activation sequences enhanced acidity has been linked to increased activation, known as the acid blob theory56,167. Therefore, phosphorylation is considered an important regulatory step in regulating Hsf1 activity. Several studies have looked into the effect of mutating a single phosphorylation site and the consequences that different stresses have on the phosphorylation pattern of Hsf165. In hHSF1, prevention of dephosphorylation increased the stability of hHSF1 trimers after heat shock and made them more transcriptionally active199. hHSF1 carries 15 of its known phosphorylation sites in its regulatory domain which corresponds to around 70% of all known phosphorylation sites. Removal of all regulatory domain phosphorylation sites resulted in a hHSF1 that still acquired nuclear localization upon stress and was able to bind DNA25. However, a lower activation threshold was seen in the phosphorylation mutant leading to a stronger response than wild type at identical temperatures25. In yeast Hsf1 substitution of 152 out of 153 serine and threonine residues with alanine residues (one serine in the DNA binding domain (DBD) was essential for function) to prevent phosphorylation resulted in attenuated Hsf1 activity at an elevated temperature205. In contrast, a higher basal and an even higher induced Hsf1 activity was achieved through phospho-mimicking substitution with aspartate residues of all serines and threonines located outside the DBD205. Taken together, a direct correlation between negative charge and Hsf1 activity was seen thus supporting the acid blob theory. The structural effects phosphorylation has on Hsf1 remain unclear but it is possible that acidification promotes a more favourable conformation for trimer stability and DNA binding. As a result, a higher degree of Hsf1 phosphorylation would increase the Hsf1-mediated transcriptional response. Phosphorylation of a protein by a kinase is a common way for cells to regulate protein activity. If a single kinase was responsible for the phosphorylation of Hsf1, chances are high that a genetic screen in yeast would have identified it. However, many kinases work on an array of targets which increases the risks of indirect effects and false positive results in mutational analyses23. Two general kinases in yeast, the PKA regulated kinase Yak1 and Snf1 a homologue of mammalian AMP-activated kinase, can phosphorylate Hsf1 in vitro69,106. In conclusion, phosphorylation of a transcription factor provides a rapid way to adjust acidification of upstream activation sequences thereby

21 controlling RNA polymerase recruitment and transcriptional activation. Thus, a model in which Hsf1 phosphorylation, carried out by multiple kinases, is an important but not an essential event in fine-tuning transcriptional activation is favoured.

HSF1 AS A CELLULAR THERMOMETER The ability of proteins to adopt different conformations makes them especially susceptible to structural changes at an elevated temperature. Hsf1 has been suggested to function as a cellular thermometer i.e. a protein that gets activated upon heat shock due to conformational changes34. In in vitro experiments monomeric mHSF1 trimerized and gained DNA binding activity at 32°C, whereas at 22°C the protein remained as inactive monomers58. Similarly, recombinant dHsf1 displayed loss and gain of DNA binding capacity when cycled between 20°C and 36°C206. Structural analyses of hHSF1 in vitro revealed unfolding of the 4th leucine zipper, a region implicated in the inhibition of trimer formation and DNA binding, upon a temperature upshift75,144. At the same time leucine zipper 1-3 were stabilized thereby resulting in increased monomer to trimer formation75. In vivo the cellular thermometer model has been tested by transfecting HSFs from other species. dHsf1 expressed in human cells was constitutively active which can be seen as a result in favour of the model since the normal temperature for a fly is more than 10°C lower than the body temperature of a human32. However, in the same study hHSF1 obtained activity in D. melanogaster already at 32°C, a stressful temperature for flies but not human cells hence arguing against a direct sensing mechanism. Moreover, hHSF1 is activated at 37°C in Xenopus oocytes, a stressful environment for frog germ cells but the basal temperature for humans210. A downside of transfection experiments is the difficulty to obtain physiological levels of the transfected protein, often the transfected proteins are severely overexpressed. Hsf1 oligomerization increases in vitro with increasing protein concentration, which in vivo could result in its activation175. In addition, if Hsf1 would be under control of a negative regulator, overexpression would titrate out its repressor and result in constitutive activation independent of temperature. Thus, it is difficult to draw any conclusions about changes in activities among HSFs when they are transfected into other organisms. Also heat shock is only one of several stressors that activate Hsf1. In conclusion, the experiments on hHSF1 in other species rather argue against a direct thermosensing by the protein and indicate that HSF activation is dependent on the state of the cellular milieu.

Figure 8. Various proposed ways of regulating Hsf1 activity. Spatial regulation (top left) by sequestration of Hsf1 from the nucleus. Activation by phosphorylation (top right). Direct sensing of an increased temperature resulting in conformational change (lower right). Titration by chaperones as a function of the amount of cellular unfolded proteins (lower left).

REGULATION OF HSF1 BY CHAPERONE TITRATION One result of Hsf1 activation is transcription of heat shock proteins (Hsps), including chaperones, many of which are essential for the cell to maintain proteostasis. Such important proteins must be under tight regulation since both too low or too high levels are toxic to the cell. A way of ensuring correct chaperone levels is through self-regulation by direct titration of a transcription factor. The idea of chaperone self-regulation was formulated before the discovery of Hsf1, influenced by experiments in E. coli, as it is one of the fastest ways to rapidly respond to an environmental signal37,147,179. Several biological processes in the cell are regulated by chaperone titration of a factor. For instance, the unfolded protein response (UPR)

23 senses proteotoxic stress in the endoplasmic reticulum (ER) and the predominant model for its regulation is based on chaperone titration where ER Hsp70 keeps the endoribonuclease Ire1 in an inactive monomeric state. Increased ER stress results in higher quantities of unfolded proteins that titrate Hsp70 away from Ire1 which then dimerizes and splices HAC1 pre- mRNA, leading to expression of the Hac1 TF that activates expression of UPR genes to alleviate the stress191. An alternative model for UPR regulation is direct sensing of unfolded proteins by Ire1, similar to the cellular thermometer model of Hsf1, resulting in HAC1 pre-mRNA splicing. In another example, the TF Pdr3 is activated by mitochondrial stress including mitochondrial import deficiencies132,193. Its latency is ensured by the Hsp70 Ssa1 which upon cytosolic mitochondrial precursor accumulation gets titrated away from Pdr3, resulting in its activation162,193. In summary, chaperone titration has been observed in various cellular compartments as a robust way to regulate transcription. Relying merely on genetic data to find regulators of Hsf1 has proven difficult. A genome-wide screen using a yeast knock-out library of single- gene deletions in which basal Hsf1 activity was measured, found that not only chaperone deletions affected Hsf1 activity22. Single deletions of genes involved in other processes also activated Hsf1, presumably because they also result in an overall increase in unfolded proteins22. This includes for example genes encoding for proteins involved in targeting proteins to other compartments such as the GET complex12. The GET complex post- translationally delivers tail-anchored proteins to the endoplasmic reticulum, hence the consequence of deleting GET1-5 is cytosolic accumulation of proteins with a highly hydrophobic, aggregation prone C-terminal domain160. Many gene deletions also result in the opposite response: lowered Hsf1 activity. These include genes involved in for example energy metabolism. Also gene deletions interfering with translation initiation, such as deletion of TIF35, results in dampened Hsf1 activity due to less newly translated proteins in need of chaperone assistance. In this situation, a high chaperone availability could potentially be the reason of inhibited Hsf1 activity. If Hsf1 is regulated by chaperone titration, then what chaperone is responsible? Deleting any gene encoding for a member of the chaperone network often results in Hsf1 activation since the output of interfering with the network is the same: an increase in unfolded proteins22,71. Most efforts have been put into determining whether Hsp70 or Hsp90 functions as a regulator of Hsf1. Both are well expressed, essential components of the chaperone network and directly involved in the folding of proteins. In yeast, they are dependent on Hsf1 for their expression even in the absence of a stressor171. Furthermore, deleting genes of Hsp70 or Hsp90 isoforms or cofactors results in the strongest misregulation of Hsf1. There are some criteria that the regulating chaperone would have to fulfil. (1) The levels of chaperone available to bind Hsf1 should be inversely

24 correlated with Hsf1 activity. (2) The chaperone should be able to interact with Hsf1 prior and after a stress. (3) During non-stressful conditions, Hsf1 should exist in a complex with the chaperone. (4) Binding affinity between the chaperone and Hsf1 should be similar to that of the chaperone and unfolded proteins. Multiple lines of evidence support the model of Hsp90 as the main regulatory chaperone of Hsf1. Depletion of Hsp90 in a cell lysate system activated hHSF1 whereas depletion of any other chaperone failed to induce hHSF1209. Moreover, chemical inhibition of Hsp90 also activated hHSF1, showing that reduced activity of Hsp90 correlated with higher activity of hHSF174,209. In Xenopus oocytes a similar activation of Hsf1 was seen upon Hsp90 depletion7. Conversely, overexpression of Hsp90 repressed mHSF1 activity204. By first depleting Hsp90 to activate Hsf1 and then adding Hsp90 back to inhibit hHSF1 again, Zou and colleagues could titrate the system both ways thereby providing compelling evidence that Hsp90 regulates Hsf1209. The next two criteria for Hsp90 to fulfil as a direct regulator of Hsf1 are the ability to interact with Hsf1 and to be able to form a complex with it under non-stressful conditions. A Hsf1-Hsp90 interaction was first observed in vitro by applying cell lysates to a column with immobilized Hsp90128. Nair and colleagues instead used recombinant hHSF1 combined with a cell lysate to see a Hsf1-Hsp90 interaction that decreased upon a 43°C heat shock129. In vivo rat HSF1 was co-immunoprecipitated with Hsp90, an interaction that was lowered upon chemical inhibition of Hsp90100. A Hsf1- Hsp90 interaction was also observed with hHSF1 in unstressed cells, an interaction that decreased upon heat shock66,209. The Hsf1-Hsp90 interaction is thought to be mediated via the regulatory domain on the Hsf1 side since regulatory domain mutants failed to form a Hsp90 complex66. Lastly, determining an affinity of Hsf1-Hsp90 and Hsp90 to unfolded proteins has not been achieved yet. Detecting a Hsf1-Hsp90 interaction without first crosslinking the cells has been challenging suggesting that Hsf1-Hsp90 may exist in a low affinity interaction complex66,209. As described earlier in the thesis, Hsp90 recognizes and binds to hydrophobic residues that can be dispersed over a large region of the client protein making affinity measurements difficult92. In summary, Hsp90 levels influence Hsf1 activity and the two proteins form a complex that dissociates upon stress. For Hsp70 to be the regulator of Hsf1, the level of Hsp70 available to bind Hsf1 should be inversely correlated with Hsf1 activity. High constitutive Hsp70 levels made rat cells recover faster from heat shock whereas reduced Hsp70 levels slowed down repression of dHsf1 activity after heat shock, suggesting a role for Hsp70 in the inhibition of Hsf1 activity after stress97. Similarly, transient Hsp70 overexpression inhibited Hsf1 activation upon stress15,165. Overexpression of Hsf1 is lethal, hence simultaneous overexpression of its inhibitor should be able to rescue

25 lethality. The minimal combination of overexpressed chaperone network proteins able to do so is Hsp70 together with a J-domain protein (JDP)205. Thus, high Hsp70 levels can prevent Hsf1 activation upon stress as well as rescue lethality from Hsf1 overexpression. Moreover, low Hsp70 levels lead to prolonged Hsf1 activation following a stress, thereby showing that Hsp70 availability affects Hsf1 activity. Hsp70 should be able to interact with Hsf1 prior and after a stress in order to act as its regulator. Hsp70 has been co-immunoprecipitated with Hsf1 by different groups in species ranging from the plant Arabidopsis to yeast and human cell lines96,165,205. In an electrophoretic mobility shift assay, addition of Hsp70 antibodies to a cell lysate incubated with a small HSE oligo resulted in slower migrating species on a gel, indicating that Hsp70 can also bind Hsf1-DNA complexes2,20. A transient dissociation of the Hsp70-Hsf1 complex was observed upon heat shock in yeast205. Furthermore, combined immunoprecipitation and mass spectrometry detected the Hsp70 proteins Ssa1 and Ssa2 as the only Hsf1 interactors205. In order for Hsp70 to meet the criteria as the Hsf1 regulator both proteins should exist in a complex during non-stressful conditions. Co- immunoprecipitation experiments described above showed an interaction and a complex, however leaving the interaction site(s) to be determined. Shi and colleagues mapped the Hsp70 binding site to the C-terminal activation domain in hHSF1165. In yeast, altering either the N- or C-terminal domain increases basal Hsf1 activity, consistent with insufficient inhibition26,131,174. Scanning deletion analysis, analysing the absence of twelve amino acid residues at the time, spanning the entire yeast Hsf1 revealed two N-terminal sites important for Hsf1 activity repression and one C-terminal site, previously defined as the CE2 domain77,81,101. Of the three sites the CE2 domain is important for Hsp70-Hsf1 interaction101. Furthermore, a mutant Hsf1 containing three CE2 domains displayed a repressed activity upon HS compared to the endogenous protein101. On the Hsp70 side, the interaction with Hsf1 is presumed to be facilitated via its substrate binding domain since ATP-treatment disassociates the complex2. These studies clearly demonstrate a Hsp70-Hsf1 complex sensitive to proteostatic perturbations. Finally, the apparent affinity between Hsp70 and Hsf1 should be similar to that of Hsp70 and unfolded proteins. Since unfolded proteins are a heterogeneous group determining a general affinity is difficult. Hsp70 recognizes short hydrophobic stretches and one can assume that depending on the folding state of the protein these hydrophobic stretches are more or less accessible. Therefore, most work on Hsp70-substrate binding has been done on shorter model peptides31. Hsf1 is a highly flexible, mostly unstructured protein that undergoes several posttranslational modifications, for instance increased phosphorylation upon heat shock, that might further affect its structure. The dissociation constant between just the Hsf1 CE2 domain and Hsp70 has been determined in vitro101. Still, determining

26 apparent affinities are probably the most difficult point to address and the chaperone field would really benefit from a reporter capable of determining Hsp70-substrate occupancy (see Concluding Remarks and Outlook for detailed discussion). To conclude, both Hsp70 and Hsp90 have been suggested as the main regulator controlling Hsf1 activity. Their increased expression result in lowered Hsf1 activity and improved stress tolerance. Conversely, lowered Hsp70 or Hsp90 levels lead to raised Hsf1 activity and decreased stress tolerance. Both Hsp70 and Hsp90 are able to interact with Hsf1 prior and after a stress. In Hsp70, a transient dissociation of the Hsp70-Hsf1 interaction have been detected. During non-stressful conditions Hsf1 has been observed in a complex with both Hsp70 and Hsp90. Hsf1 interaction with Hsp70 appears to be of a higher affinity since it can be readily detected and does not require addition of recombinant protein, which was needed in some Hsp90 studies to detect a Hsf1-Hsp90 interaction. Since Hsp70 and Hsp90 both have a key role in de novo folding of proteins it is difficult to separate interactions occurring during Hsf1 biogenesis with interactions regulating Hsf1 activity. As a final point, the highly flexible structure of Hsf1 and the heterogeneous composition of unfolded proteins complicates affinity measurements to Hsp70 and Hsp90. Moreover, depending on the multimeric state of Hsf1 and the amount post-translational modifications it is subjected to further affinity changes might occur. To date, it is still debated if Hsp70 or Hsp90 is the main regulator of Hsf1 activity.

27 METHODOLOGY Science depends on precise and reliable methods in order to facilitate new discoveries. It is of great importance that new data can be attributed as a bona fide discovery and not a result of variations due to imprecise methods. The available methodology has helped researchers gain a deep understanding how cells ensure proteostasis through transcriptional regulation. However, methods to determine the size of the available pool of chaperones at a given time is missing. Such tool would allow researchers to study how cells allocate their chaperone resources and how chaperone availability affects Hsf1 activity.

METHODS TO STUDY HSF1 ACTIVITY The chaperone field has been intensively studied for almost half a century and Hsf1 was one of the first well-characterized transcription factors (TF), making studies of its regulation a paradigm for understanding transcription- factor regulation. Hsf1 has one major role in the cell namely to bind heat shock responsive elements (HSE) upstream of genes to activate transcription. Early experiments used genomic footprinting, a method to study protein-DNA interactions in living cells to understand how Hsf1 binding to DNA was influenced by different environmental conditions3,176. Once HSEs were established as the binding sites electrophoretic mobility shift assays were employed using short specific DNA probes together with cell lysates to determine Hsf1-HSE binding under diverse conditions3,175,209. By incorporating HSEs into minimal promoters driving a reporter gene, e.g. β-galactosidase, Hsf1 activity could be measured for the first time169. As HSEs were detected in many genes encoding heat shock proteins (Hsps), increased proteins levels determined by western blot were also used as a measurement of Hsf1 activity. Before real time PCR could quantitatively detect transcript levels, a combination of PCR and northern blotting was used to visualize changes in transcript levels upon Hsf1 activation200,201. Together with mutational analyses of both Hsf1 and HSEs the above methods helped the field gain insight into Hsf1-activity regulation. More recent techniques include chromatin immunoprecipitation (ChIP) where promotor occupancy by a given protein can be determined and various sequencing methods such as mRNAseq, ChIPseq and NETseq30,85,192. mRNAseq provides quantification of all mRNAs in the cell possibly resulting in over- and under representation of stable and unstable mRNAs respectively. NETseq gives information about nascent mRNAs still associated with the RNA polymerase thereby providing a snapshot of what genes are transcribed at a given time without being affected by mRNA stability. Together, these two methods have provided information about transcriptional changes upon various stresses which also involves other TFs.

28 ChIPseq extends the ChIP technique to include the whole genome which has been valuable in order to identify novel Hsf1-regulated genes and to assess the impact Hsf1 has on gene regulation at various conditions.

METHODS TO STUDY HSP70 AVAILABILITY Hsp70 plays a role in numerous cellular processes related to protein folding, remodelling and degradation. This gives rise to many ways to measure Hsp70 availability since failure or reduced capacity to perform a certain process can be seen as a readout for a Hsp70 deficiency. However, those will only be indirect measurements since the cause of the defect might as well be due to the lack of a cofactor involved in the process. Indirect ways to measure Hsp70 availability include assessing the folding of a newly synthesized protein or determining degradation rates of a misfolded model substrate. Destabilized versions of firefly luciferase that form foci upon proteotoxic stress have been developed as proteostasis sensors, as an indirect measure of a chaperone deficiency67. Moreover, since Hsp70 aids in translocation of proteins across membranes, accumulation of mitochondrial precursor proteins or accumulation of proteins destined for the ER in the cytosol are indicators of a Hsp70 deficiency. A direct association between Hsp70 and Hsf1 makes Hsf1 activity, i.e. induction of the heat shock response, a good, but still indirect way of measuring Hsp70 availability since transcriptional activation depends on recruitment of a number of downstream factors such as RNA polymerase. Furthermore, the fact that Hsp70 is merely one component in a network of proteins involved in proteostasis maintenance makes it difficult to directly measure its availability even though the specific short hydrophobic stretch it recognizes could prove helpful. To date a direct way of measuring Hsp70 availability e.g. using a reporter, is missing and further efforts should be allocated at developing such a method.

30 AIMS OF THIS THESIS

The overall aim of this thesis is to understand how cells control protein homeostasis through transcriptional regulation and to develop new methods to promote further discoveries in this field. Specifically, I have addressed the following aims:

Aim I: Elucidate how cytosolic and nuclear pools of available Hsp70 regulate Hsf1 activity.

Aim II: Determine the role of the cytosolic splice isoform Fes1S in controlling proteostasis and Hsf1 latency.

Aim III: Characterize the luciferase NanoLuc as a transcriptional reporter for the fast kinetics of Hsf1 activity upon stress in yeast.

Aim IV: Describe how NanoLuc luciferase can be utilized as a co-translational folding reporter to monitor proteostasis.

Aim V: Reveal how mitochondrial translation efficiency impacts on cytosolic proteostasis.

32 SUMMARY OF INDIVIDUAL STUDIES

STUDY I Aim I: Elucidate how cytosolic and nuclear pools of available Hsp70 regulate Hsf1 activity. Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing genes that restore perturbed proteostasis. In budding yeast, Hsf1 is constitutively nuclear and Hsp70 is a prime candidate for the stress- titratable repression of its activity. The chaperone-titration mechanism was investigated by reconstituting and characterizing the large Hsf1-Hsp70 complexes in vitro. In vivo data demonstrated a direct interaction between the substrate binding domain (SBD) of Hsp70 and Hsf1, as well as highlighted that newly translated proteins misfold and titrate Hsp70 during heat shock. We also showed that the regulatory interactions between Hsf1 and Hsp70 are controlled by the nuclear levels of nucleotide exchange factors (NEFs). Completely unleashing Hsf1 from Hsp70 repression revealed a cryptic genetic hyper-stress program with a widely broadened gene-target signature and dramatically amplified transcriptional effects.

RESULTS AND DISCUSSION The Hsf1-Hsp70 complex can be formed in vitro and in vivo In order to directly test if Hsp70 could regulate Hsf1 we used a dual step purification protocol to purify the complex in vitro. Functional testing of the purified proteins revealed a large 620 kDa complex with DNA binding capacity. Further Hsp70 binding to the complex reduced its affinity for DNA. ATP addition caused dissociation of the complex indicating that Hsf1 was bound by the SBD of Hsp70. We set out to directly test this in vivo by utilizing a crosslinking method. Strikingly, the SBD contacted Hsf1 under non-stressful conditions. Upon heat shock the interaction weakened probably because of increased competition for the SBD with unfolded proteins. Thus, Hsp70 regulates Hsf1 activity by binding via its substrate binding domain.

Genetic alterations of Hsp70 co-factors reveal spatial regulation of the Hsf1-Hsp70 interaction NEFs aid in the removal of ADP from Hsp70 so that ATP can re-bind thereby lowering the affinity Hsp70 has for unfolded proteins or Hsf1. We overexpressed the NEF Sse1 either in the cytosol or nucleus to enhance the

33 rate at which Hsp70 releases substrates. Surprisingly, cytosolic overexpression of Sse1 had no effect on Hsf1 activity while nuclear overexpression greatly enhanced Hsf1 activity both under basal and induced stress conditions. Thus, these experiments uncovered a spatial regulation of Hsf1 where cytosolic and nuclear pools of Hsp70 are not readily exchangeable. Next, we chose an opposite approach in which we inactivated cytosolic Hsp70 by limiting its ability to release substrates, thereby making it unable to titrate Hsf1 in the nucleus. This was done by deletion of FES1 which resulted in strong constitutive Hsf1 activity under all conditions tested. Transcriptional profiling of WT and fes1Δ cells under non-stressful conditions and after a transient heat shock revealed that Hsp70 inactivation by unfolded proteins resulted in a broadened stress response. Specifically, many Hsf1-target genes displayed hyper-induced characteristics possibly by additional recruitment of Hsf1 to low affinity binding sites. Taken together, the genetic modifications affecting Hsp70 substrate release exposed a spatial regulation of Hsf1 activity where severe out-titration of Hsp70 triggers a genetic hyper-stress program.

Newly synthesized proteins titrate Hsp70 away from Hsf1 upon stress Unfolded or misfolded proteins are the factor competing for Hsp70 SBD binding with Hsf1 but the origin of these proteins remains unclear. The prime suspects were newly synthesized proteins that have not yet achieved their final native fold. Therefore, we tampered with the available pool of newly synthesized proteins. We either increased the pool by addition of a toxic amino acid analogue or decreased the pool through lower translational rates caused by amino acid starvation or peptide-elongation blockage by cycloheximide. An increase in newly synthesized proteins unable to achieve their final fold efficiently activated Hsf1 and resulted in additional activity when combined with a heat shock. In contrast, we observed that lowering the amount of newly synthesized proteins in the cell led to a significantly attenuated Hsf1 activity. These data point to newly synthesized proteins as the proteins that primarily misfold upon stress, hence competing with Hsf1 for available Hsp70. In summary, in study I we showed that Hsp70 binds Hsf1 through its SBD. Also, our data underline that the nuclear pool of free Hsp70 tightly controls Hsf1 DNA-binding capacity and represses a genetic emergency program that is triggered by proteostasis collapse.

34 STUDY II Aim II: Determine the role of the cytosolic splice isoform Fes1S in controlling proteostasis and Hsf1 latency. Fes1 is a co-factor of Hsp70 that aids in substrate release from Hsp70 both by facilitating nucleotide exchange and by direct competition with substrates. Here, we have investigated a rare event of alternative splicing in yeast resulting in two FES1 isoforms, Fes1S and Fes1L. Fes1S is localized to the cytosol whereas Fes1L in targeted to the nucleus. In vitro measurements demonstrated that both isoforms carry similar NEF activity. We show that Fes1S is required for growth at elevated temperatures as well as for the turnover of previously known Fes1-dependent substrates. Fes1S is upregulated by heat shock and also the isoform required to keep Hsf1 repressed. Overexpression of Fes1L as the only FES1 isoform can rescue all phenotypes associated with Fes1S except repression of Hsf1 activity.

RESULTS AND DISCUSSION Splicing of FES1 transcripts results in the isoforms Fes1S and Fes1L that localizes to the cytosol and nucleus respectively We found that the FES1 pre-mRNA undergoes alternative splicing resulting in two functional isoforms: Fes1S and Fes1L. The isoforms arise as a result of competition between polyadenylation and splicing machineries. Splicing results in translation of Fes1L carrying a putative nuclear localization sequence (NLS) and our data showed that it was targeted to the nucleus whereas Fes1S remained in the cytosol.

Fes1S is essential to repress Hsf1 activity under non-stressful conditions Our lab has previously discovered that Fes1 is involved in turnover of certain misfolded-model proteins, thermotolerance and repression of Hsf1 activity62. Here, we found that these processes depend on the major Fes1 isoform Fes1S. Intriguingly, overexpression of Fes1L complemented both protein turnover and supported cellular growth at higher temperatures in a fes1ΔS background. However, despite taking care of the proteostasis problem it was not able to repress Hsf1 activity. Taken together, study II identified a rare example of alternative splicing that result in the expression of two functional isoforms of Hsp70 NEF Fes1: Fes1S and Fes1L. Fes1S localizes to the cytosol whereas Fes1L is targeted to the nucleus. Fes1S is involved in maintaining protein homeostasis by releasing misfolded proteins from Hsp70 to facilitate their degradation thereby increasing Hsp70 availability, thus functioning as a negative regulator of Hsf1 activity.

35 STUDY III Aim III: Characterize the luciferase NanoLuc as a transcriptional reporter for the fast kinetics of Hsf1 activity upon stress in yeast. Here, we report on a new method developed for using the 19 kDa protein luciferase NanoLuc in yeast. Other reporters traditionally used to study Hsf1 activity, such as β-galactosidase or GFP, take time to translate and fold making real time activity assessments impossible. Instead, these reporter kinetics often show a delayed activation signal and lack of repression due to the long half-lives of the used reporter proteins. This study characterizes NanoLuc, codon optimized for yeast expression (yNluc), and its ability to report on rapid transcriptional changes as well as its function as an activity tag to monitor protein turnover.

RESULTS AND DISCUSSION Codon optimized genes expressing yNluc with or without a destabilizing PEST sequence did not cause any toxicity in yeast as determined by growth on plate and in liquid culture. The reporters showed great sensitivity, with yNluc expressed from a minimal heat-shock responsive promoter recognizing as little as 1000 cells expressing basal Hsf1 activity. We assessed the ability of yNluc under the same promoter, carrying a PEST degron for increased turn-over to report on the rapid changes in Hsf1 activity upon a transient heat stress. Strikingly, simultaneous monitoring of mRNA levels and protein levels revealed that the bioluminescent signal faithfully reported on the mRNA levels. In addition, C-terminal fusions of yNluc to a protein of interest could be utilized to monitor steady state levels as well as protein turnover. In conclusion, in study III we showed that yNluc and yNlucPEST are functional reporters in yeast that due to their small size and ease of use provide a valuable tool to monitor Hsf1 activity and protein levels in the cell.

36 STUDY IV Aim IV: Describe how NanoLuc luciferase can be utilized as a co-translational folding reporter to monitor proteostasis. Proteins need to achieve their native three-dimensional structure in order to be physiologically active. Similarly, reporter proteins will only gain their reporting function if they are correctly folded. As reported in study III, yNluc is a protein that faithfully reports on transcriptional changes through its bioluminescent signal in WT cells. Here, we make further use of yNluc by utilizing it as a reporter for co-translational folding. We show that upon proteostatic stress newly synthesized yNluc fails to fold into its native conformation thereby providing a novel tool to directly monitor de novo protein folding.

RESULTS AND DISCUSSION We assessed the function of yNluc driven by a minimal reporter containing Hsf1 binding sites in WT and a proteostasis mutant. In WT cells yNluc was induced upon heat shock in terms of transcripts, protein and bioluminescent signal. In a proteostasis mutant a similar but stronger effect was observed except for a decrease in bioluminescent signal consistent with a de novo protein folding defect upon stress. Constitutive yNluc expression during a heat shock remained stable in WT and mutant strains revealing that it is not unfolding of already native protein but instead problems with de novo protein folding. Finally, 17 stabilizing mutations were introduced in yNluc to create yeast super folded NanoLuc (ysfNluc) which further enhanced folding of the protein in a WT scenario upon stress whereas the proteostasis mutant remained unaffected. In summary, yNluc and ysfNluc represent two novel reporters that, for the first time allows real time measurements of de novo protein folding upon changes in the cellular milieu.

37 STUDY V Aim V: Reveal how mitochondrial translation efficiency impacts on cytosolic proteostasis. Cells are dependent on mitochondria for the production of ATP as well as for regulating calcium homeostasis, metabolism and apoptosis. Mitochondria carry a small genome that is transcribed and translated in the organelle. Mitochondrial translation closely resembles bacterial translation and based on this we constructed two mutants, each carrying one individual point mutation in the accuracy centre of the mitochondrial ribosome. The mutant cells displayed either hypo- or hyperaccurate mitochondrial translation and were evaluated for cytosolic proteostasis capacity and longevity. Hypoaccurate mitochondrial translation resulted in increased oxidative stress upon ageing that had adverse effects on chronological lifespan. In contrast, hyperaccurate mitochondrial translation had a beneficial effect on cytosolic proteostasis and prolonged lifespan. This shows that mitochondrial proteostasis directly affects cytosolic proteostasis through inter-organellar crosstalk.

RESULTS AND DISCUSSION Hyperaccurate mitochondrial translation improves cytosolic proteostasis We assessed the effects hypo- or hyperaccurate mitochondrial translation had on cytosolic proteostasis. After a heat shock both WT cells and the mitochondrial translation mutants showed equal levels of cytosolic Hsp104 decorated foci. However, in the hyperaccurate mutant aggregates got cleared at a significantly faster rate than in WT and in the hypoaccurate mutant which both cleared aggregates at the same rate. The improved cytosolic capacity to buffer proteostatic perturbations was also seen in terms of refolding rate of heat denatured cytosolic firefly luciferase. Both these processes require Hsp70 and can thereby be viewed as a measurement of available cytosolic Hsp70. Another Hsp70-mediated process is protein turnover via the ubiquitin proteasome system (UPS). Strikingly, the hyperaccurate mutant displayed an increased turnover of R-GFP, an N-end rule model substrate. Furthermore, the toxic misfolded cytosolic protein ΔssPrA that is targeted for UPS-dependent nuclear degradation was less toxic in the hyperaccurate mutant compared to WT. Thus, mitochondrial hyperaccurate translation is giving rise to an extended cytosolic capacity of the proteostasis system through more available Hsp70.

Hypoaccurate mitochondrial translation triggers expression of an interorganellar proteostasis transcription program (IPTP) In order to figure out how nuclear transcription is affected by hypoaccurate mitochondrial translation we performed RNAseq on WT cells and the two

38 mutant strains during logarithmic growth and post diauxic shift i.e. after the shift to a non-preferred carbon source. While a majority of the transcriptome remained the same during logarithmic growth the hypoaccurate mutant induced IPTP post diauxic shift. Characterization of this novel transcriptional program revealed an induction of genes important for mitochondrial proteostasis, protein import and mitochondrial translation. Strikingly, direct testing of selected genes that were induced revealed that their induction depended on Msn2/4 and that failure to induce the IPTP transcriptional program resulted in decreased longevity. In short, study V showed that organellar proteostasis impacts on cytosolic proteostasis. Specifically, it elucidates how mitochondrial translation accuracy affects availability of cytosolic Hsp70.

40 CONCLUDING REMARKS AND OUTLOOK

The work presented in this thesis has aimed at elucidating how cells control proteostasis through the regulation of Hsf1 using yeast as a model system. The work sheds light on how nuclear and cytosolic pools of available Hsp70 play distinct roles in maintaining Hsf1 latency and contribute to cellular proteostasis. Additionally, novel methodology to monitor proteostasis and Hsf1 activity have been developed in order to facilitate further discoveries. As many components of the proteostasis system studied here are conserved from yeast to man, lessons learned from yeast provide an excellent foundation to understand how multicellular organisms handle proteostatic perturbations caused by for example ageing and disease. In the first study a spatial regulation of the constantly nuclear Hsf1 by distinct nuclear and cytosolic Hsp70 pools was proposed. Nuclear J-domain proteins (JDPs), for example Sis1, have already been discovered thereby prompting the requirement for a nuclear nucleotide exchange factor (NEF) in order to enable efficient Hsp70 cycling of substrate binding and release. This NEF was identified as Fes1L in our second study where we show that alternative splicing resulted in the addition of a nuclear localization sequence to the protein. Overexpression of the nuclear Fes1L in a Fes1S deletion background could complement both the temperature sensitive phenotype as well as turnover of misfolded proteins. Surprisingly, no suppression of Hsf1 activity was seen even though Fes1L overexpression alleviated the protein folding stress. In light of more recent data from study I revealing that overexpression of Sse1 in the nucleus activated Hsf1 despite no apparent proteostatic defect, the Fes1 model can be extended. Likely, Fes1L overexpression resulted in a spill over of the protein to the cytosol. Presumably this was enough to maintain enough available Hsp70 to complement the first two phenotypes observed. On the other hand, increasing the nuclear pool of Fes1L likely enhanced Hsf1 release from Hsp70, in a similar manner as nuclear targeted Sse1, thereby not providing any suppression of the heat shock response (HSR). Furthermore, in our second study it was observed that Fes1S was the isoform mainly produced upon heat shock. Impaired processing of mRNA transcripts by the spliceosome has previously been observed in D. melanogaster as a result of chaperone deficiency during and after a severe heat shock202. This could

41 explain the data even though the heat shock was mild and therefore perhaps did not titrate away all available chaperones. Another model explaining the data is that increased Fes1S protein levels is an adaptation to ensure efficient Hsp70 cycling when the burden of unfolded newly synthesized proteins increase in the cytosol. Further investigations should be aimed at explaining how cytosolic and nuclear pools of Hsp70 are regulated as well as how communication between the two pools are achieved. A direct way of determining the available pool of Hsp70 in cells at a given time has not yet been discovered. Tampering with levels of Hsp70 cofactors provides an indirect way of titrating the available pool of Hsp70. Yet, a direct assay for measuring the levels of free versus substrate-bound Hsp70 is missing. Protein-fragment complementation assays such as split-ubiquitin or split-GFP assays are valuable tools to detect protein-protein interactions but suboptimal for transient interactions since they are not reversible53,86. Split-firefly luciferase provides a reversible reaction with a real-time bioluminescent readout116. However, firefly luciferase is sensitive to proteostatic perturbations and depends on Hsp70 for its folding159. NanoBiT, a Nanoluc luciferase split into an 18 kDa and 1.3 kDa domain could provide a good starting point due to its small size and no reported chaperone dependency for its folding38. By fusing one part to Hsp70 and another part to a small hydrophobic peptide containing one or more Hsp70-recognition site(s), a possible direct readout of Hsp70 availability could be obtained. For instance, a high bioluminescent signal would indicate low competition for the peptide with other unfolded substrates whereas a lower signal would be consistent with higher competition for Hsp70 binding. Still, the reduced bioluminescent signal in proteostasis-network mutants compared to WT cells upon stress reported in study IV is worrisome from a methodological point of view but should not provide a problem in WT cells. A reporter able to measure Hsp70 availability would provide the chaperone field with invaluable information about how cells allocate their chaperone resources not only during low and high levels of proteotoxic stress but also during ageing and disease. Cytosolic Hsp70 availability was shown in our final study to depend on mitochondrial translation efficiency where improved translational accuracy positively correlated with Hsp70 availability in post-diauxic cells. Hypoaccurate mitochondrial translation resulted in a specific transcriptional response denoted IPTP with a strong induction of e.g. Hsp70, Hsp90 and their cofactors that to some but not full extent was a result of increased Msn2/4 activity. Instead, a co-operative effort by Msn2/4 and Hsf1 is possible as many members of the Hsp70 family carry both responsive elements. Also, other transcription factors are likely to be involved. Previous studies have linked a temperature sensitive allele of HSF1, mas3, to impaired mitochondrial import170. mas3 contains a stop codon in its trimerization domain and is defective in SSA1 and SSA3 induction thereby resulting in a

42 cell with greatly reduced Hsp70 availability170,184. Although no import defect was observed for the hypoaccurate translation mutant in our study during logarithmic growth it has not been tested whether is arises later together with all other cytosolic proteostasis defects observed. IPTP induction may therefore be a result of a Hsp70 deficiency due to a combination of increased accumulation of proteins destined for the mitochondria in the cytosol and a deficit of the same proteins in the mitochondria. More work directed at investigating the potential role of Hsf1in the IPTP response should be carried out. Hsf1, also known as “the master regulator of the heat shock response” is together with the chaperones Hsp70 and Hsp90 a putative drug target in several diseases e.g. cancer, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis etc. Hsp90 has, due to its ability to stabilize proteins a role in buffering mutations thereby preventing proteins carrying destabilizing mutations from degradation110. This effect has been observed in cancer where Hsp90 buffering has enabled secondary stabilizing mutations to occur which in turn could contribute to malignancy. Hsf1 activity is upregulated in many cancers as rapid cellular growth with elevated translational rates requires high levels of chaperones. In addition, Hsf1 drives a transcriptional program that is distinct from the one observed in cells upon heat shock which is linked to malignancy and associated with decreased survival rates in humans35,122. Increased levels of Hsp70 serves to repress apoptotic signals hence enabling further growth and division of malignant cells. In contrast to cancer, high chaperone levels are beneficial for treatment of Alzheimer’s and Parkinson’s disease where aggregated proteins result in neuronal death. An increased level of chaperones could act on the misfolded and aggregated proteins to target them for degradation thereby reducing toxicity. Cystic fibrosis provides a third example of how controlling chaperone levels can provide a treatment strategy114. By lowering chaperone levels in the endoplasmic reticulum (ER) the disease-causing destabilized ion channel could escape ER-associated degradation, thereby reaching its final destination in the plasma membrane. Even though the ion channel has defects many identified mutants still carry some activity which could ease many of the symptoms. These examples highlight the importance of studies regarding cellular proteostasis at a basic molecular level. By understanding the roles of each component in the proteostasis network and how they are regulated, proteostasis-targeted therapies can be developed to combat a number of diseases.

44 ACKNOWLEDGEMENT

Many people have contributed directly or indirectly to this thesis and for that I am immensely grateful. I would especially like to thank: My supervisor Claes Andréasson, you taught me how to actually think and be (overly) critical. I will never forget how I stepped out your office after yet again hearing that you couldn’t afford another PhD student. However, a few minutes later you followed me into the lab and for some reason you had changed your mind. Thank you so much for giving me the chance and supervising me these 5 years. I did not know it when I started but pretty soon I realized that I picked one of the best! Marie Öhman for having been a very supportive co-supervisor through seminars, follow-ups and 5k races around Stora Skuggan. The Andréasson group for all the good times both inside and outside of the lab. Naveen, thank you for being the funny guy and for setting a very high standard for the rest of us students. Thank you Nandu for teaching me about how to season my food beyond salt and pepper. I have had a lot of fun in the lab and when going to conferences with you. Ganapathi, thank you for assisting me with the first NanoLuc paper. Melania, finally some female company! Thank you for bringing our lab into order. Joydeep and Jany, I am very curious about how your stories will develop. Thank you Joydeep for the scientific discussions and good luck with your PhD Jany. I would also like to thank past and present students for injecting some new energy into the lab. It is always interesting to have you around. Our collaborators, together we have reached a better understanding about our scientific questions! Thank you Wenjing, Marc Friedländer, Tamara, Hanna, Martin Ott, Carlotta, Sabrina Büttner and all the other people who contributed to the studies included in this thesis. I would like to thank the Ljungdahl, Ott, Büttner and Stelkens groups for your scientific input and nice discussions during our yeast meetings. Many thanks to the “old” cellbio people that welcomed me to the corridor especially Misa and Matthias. Roger Karlsson, the opportunity to do my master thesis in your lab changed my life on so many levels. The extended F4 corridor with all its people, thank you for making this a great workplace. Thank you Deike, Kicki, Steffi, Anna V, Bejan and Mahsa for brightening my morning cup of tea. Also, thanks to past and present members of the Jonas group for all the food workshops and parties!

45 Slutligen, för jag har sparat de allra bästa till sist, vill jag tacka min familj. Tack mamma och pappa för att ni ALLTID stöttar mig, oavsett vad det gäller. Ni har lärt mig så mycket av det som verkligen spelar roll, som att äta med bestick och att läsa. Jag hoppas att denna avhandling kan ge er lite klarhet i vad jag faktiskt har sysslat med dessa 5 år. Tack mormor och farmor för att ni fortfarande finns här för mig. Ni har gett mig fantastiska barndomssomrar, stickade strumpor och tröjor i massor men framförallt ert fulla stöd (och mat). Deike, känslan av att ha någon nära som vet vad doktorsstudier innebär är ovärderlig. Tack för all hjälp och känslomässigt stöd i arbetet med denna avhandling och i det verkliga livet.

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