Proteostasis is a network that insures the proper folding, degradation, and trafficking of proteins. Metacaspases have essential role in programmed cell death and other processes that help cell sustain life. For instance, the Saccharomyces cerevisiae metacaspase 1, ScMCA1, regulates insoluble protein aggregates which promote cellular fitness. Previously, our group reported that different vacuolar catabolism proteins such as Lap4, are altered in the absence of ScMCA1. Lap4 (Vacuolar aminopeptidase 1) plays a major role in yeast's selective autophagy pathway to degrade unwanted materials. Additionally, they reported alternations of heat shock proteins, specially Hsp12 in ΔScMCA1 strain. Therefore, we hypothesised that elevation of Hsp12 and Lap4 expression that leads to vacuolar increase and chaperone activity may serve as compensatory mechanisms to manage the clearance of insoluble protein aggregates when ScMCA1 is lost or impaired. Our results support this theory by showing high expression level of Hsp12 and Lap4 in absence of ScMCA1. We observed high insoluble protein aggregation accumulation in ΔScMcA1 strain lacking Lap4/or Hsp12 compared to wildtype (WT), specially when they are stressed. Those strains also exhibited robust vacuolar response possibly to degrade insoluble protein aggregates through autophagy action. As a result, these alternations in proteostasis may be responsible for decreased cell viability observed in ΔScMCA1+ΔLap4 and ΔScMCA1+Hsp12 under stress conditions.

In addition to apoptosis, metacapases function to regulate various other processes that promote and sustain life. For example, the Saccharomyces cerevisiae metacaspase Yca1 promotes cellular fitness by regulating insoluble protein levels. However, the mechanism(s) that regulate this proteostasis function for Yca1 have remained elusive. Here, using proteomics coupled to protein interaction studies, we describe a role for Yca1 in restraining deposition to the insoluble proteome and further identify a post-translational regulatory mechanism for modulating Yca1 function. Our initial analyses uncovered a role for Yca1 in aggregate assembly where Yca1, in coordination with the Cdc48 chaperone, regulates the composition of the insoluble proteome. Interestingly, loss of Yca1 was correlated with reduced sequestration of proteins related to ribosomal and translational processes in the insoluble protein fraction during heat stress. Subsequent proteomic analyses identified a regulatory mechanism for Yca1 mediated by the ubiquitin system, a feature that was instrumental for limiting insoluble protein content. Specifically, we noted K355 ubiquitination and S346 phosphorylation as key modifications that directed Yca1 function to maintain proteostasis. Loss of function mutations at these sites led to increased retention of insoluble protein and increased vacuolar structures. Surprisingly, loss of Yca1 also affected ubiquitin homeostasis in vivo as observed by reduced levels of low molecular weight free ubiquitin. Upon further analysis, we observed that the ubiquitin precursor protein Rsp31 was cleaved by Yca1 suggesting a possible role for Yca1 in de novo ubiquitin synthesis. Together, these analyses suggest that post-translational modifications of Yca1 are critical regulatory features for this protease, and that this enzyme regulates cell proteostasis in combination with other chaperone and protein degradation machinery.

Ankyrin-B (Ank-B) is a crucial scaffolding protein regulating expression and localization of contractile machinery in the cardiac muscle. Recent genetic investigations in the First Nations Community, the Gitxsan of Northern BC, identified a mutation in Ank-B (p.S646F c.1937 C>T) associated with a cardiac arrhythmia, Long QT Syndrome Type 4 (LQTS4). Distinct from other LQTS4 subtypes, individuals harbouring the p.S646F variant exhibit development deficits including cardiomyopathies and accessory electrical pathways. How p.S646F interferes with the development of the heart is unknown due to a fundamental lack of understanding regarding Ank-B proteostasis and its role in cardiac differentiation. Initial in silico analyses predicted the p.S646F mutant to be deleterious to the Ank-B protein. Using in vitro techniques, I determined p.S646F mutant reduced levels of Ank-B in H9c2 rat ventricular cardiomyoblasts. Furthermore, haploinsufficiency in mice was previously shown to result in developmental cardiac deficits. I, therefore, hypothesized that p.S646F interferes with Ank-B proteostasis, thereby affecting cardiomyocyte development. I showed that p.S646F destabilized Ank-B in cardiomyoblasts, due to increased degradation via the proteasome. Furthermore, overexpression of p.S646F Ank-B had a significant impact on cellular behaviour including reduced cell viability, and altered expression of cellular differentiation markers. Together these data address critical knowledge gaps with regards to Ank-B protein homeostasis and the role of Ank-B in cardiomyocyte viability and development. These findings inform the diagnosis and treatment of patients with the p.S646F variant, creating potential targeted pathways of intervention, and furthering our understanding of the role of the Ank-B in the development of the heart. / Graduate / 2019-04-26

Le mélanome cutané est un cancer très agressif, responsable de 80% des décès liés aux cancers de la peau. Le mélanome métastatique (MM) est souvent résistant à la radiothérapie et aux chimiothérapies. Sa progression est majoritairement initiée par des mutations oncogéniques des gènes BRAF et NRAS activant la voie de prolifération MEK/ERK. Le MM est difficile à traiter malgré le succès de nouveaux traitements (thérapies ciblant l’oncogène BRAFV600E et immunothérapies), qui sont cependant limités à certains patients. De plus l'émergence de résistances ne permet pas d’obtenir une réponse durable, ce qui incite à rechercher de nouvelles cibles tumorales. Dans les cellules cancéreuses, l’accumulation d’altérations génétiques et le fort index prolifératif accroissent leur addiction aux mécanismes de contrôle de la qualité du protéome, comme le système ubiquitine-protéasome (UPS). L’UPS comprend une machinerie protéolytique (le protéasome 26S) et un réseau d’enzymes régulant l’ubiquitination de protéines cibles. La réaction enzymatique de retrait de l’ubiquitine est la déubiquitination, réalisés par de protéases spécifiques appelées DéUBiquitinases (DUBs). Malgré l’importance des DUBs dans de nombreuses situations pathologiques comme le cancer, leur implication dans la physiopathologie du mélanome est mal connue. Afin d’identifier des DUBs dont l’activité est modulée dans le mélanome, nous avons utilisé une méthode d’étiquettage biochimique in vitro des DUBs actives (‘’DUB trap assay’’) qui nous a permis d’identifier USP14 (Ubiquitin Specific Protease 14) dont l’activité est augmentée dans nos lignées de mélanome par rapport aux mélanocytes. USP14 est associée physiquement au protéasome, avec un rôle important sur la protéostasie cellulaire en général. L’analyse de données bioinformatiques publiques confirme l’importance de USP14 dans le mélanome en associant l’expression du gène USP14 à la progression du mélanome et à un mauvais pronostic. Nous avons ensuite montré que cibler USP14 par des approches génétique (siRNA) ou pharmacologique (inhibiteurs de l’activité) a un effet anti-mélanome in vitro et in vivo, associé à une accumulation de protéines polyubiquitinées, générant un stress du réticulum endoplasmique, la dépolarisation de la mitochondrie et une production de ROS, aboutissant à une mort indépendante des caspases. Cet effet cytotoxique est obtenu indépendamment du statut mutationnel des protéines oncogéniques (BRAFV600E, NRAS, NF1), des suppresseurs de tumeurs (TP53, PTEN), du niveau de résistance aux thérapies ciblées ou du statut phénotypique des mélanomes. Ces résultats indiquent que USP14 représente une nouvelle cible thérapeutique pertinente dans le mélanome. Dans la continuité de ces travaux, j’ai cherché à identifier d'autres DUBs pouvant jouer un rôle dans la prolifération et la survie des cellules de mélanome en réalisant le criblage d'une banque de siRNA ciblant 90 DUBs sur une lignée de cellules de mélanome. Outre le fait de confirmer l’implication de USP14 dans la prolifération du mélanome, ce criblage génétique révèle que la déplétion d’une autre DUB associée au protéasome a un puissant effet antiprolifératif sur les cellules de mélanome. Nos travaux préliminaires montrent que le ciblage de cette nouvelle DUB se traduit par un arrêt de prolifération suivi d’une mort cellulaire associée à des dommages à l’ADN in vitro et in vivo. Dans l’ensemble, mes travaux de thèse révèlent un rôle essentiel des DUBs associées au protéasome dans la prolifération et la survie du mélanome, et ouvrent la piste à de nouvelles stratégies thérapeutiques ciblant les mécanismes aberrants de la protéostasie tumorale de ce cancer. / Non communiqué

The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Les cellules sont constamment exposées à des stress « protéotoxiques » qui altèrent leurs protéines. Si les protéines endommagées ne sont pas réparées ou éliminées, elles peuvent former des agrégats toxiques pouvant conduire à l’émergence de plusieurs maladies, telle que les maladies neurodégénératives et le cancer. Pour éviter cette toxicité, les cellules ont développé plusieurs stratégies qui collaborent et communiquent afin d'assurer le contrôle de qualité des protéines et maintenir l’intégrité du protéome cellulaire. L’ensemble de ces stratégies forment le réseau de l’homéostasie protéique ou « protéostasie ». Ce réseau inclus les chaperonnes moléculaires, les systèmes protéolytiques (lysosomes, protéasomes) et des systèmes de séquestration des protéines endommagées. L’Ubiquitine et les protéines apparentées à l’Ubiquitine telle que SUMO et NEDD8, sont des effecteurs essentiels de ce réseau. Ces molécules modifient leurs substrats de façon covalente, grâce à l’action d’une cascade d’enzymes E1, E2 et E3. En principe, on considérait que chacune de ces voies employait sa propre cascade enzymatique pour la modification post-traductionnelle de ses substrats. L’Ubiquitination joue un rôle essentiel dans la réponse au stress cellulaire, surtout en assurant la dégradation protéasomique des protéines mal repliées. Récemment, notre laboratoire a trouvé que plusieurs stress protéotoxiques telle que l’inhibition du protéasome, un choc thermique et un stress oxydatif, causent une augmentation de NEDDylation. De manière remarquable, cette augmentation ne dépend pas de l’enzyme d’activation de NEDD8 NAE, mais plutôt de celle de l’Ubiquitine Ube1. De plus, elle se caractérise par la formation des chaînes poly-NEDD8 et des chaînes mixtes entre NEDD8 et Ubiquitine. Ce processus est réversible et une restauration cellulaire est obtenue une fois le stress atténué. Le but de notre projet est de caractériser la réponse de NEDD8 au stress cellulaire ou ce qu’on appelle « la NEDDylation atypique » en vue de comprendre son effet biologique pendant ces conditions. Nos résultats montrent que la NEDDylation atypique dépend des protéines de stress Hsp70/90 et qu’elle cible principalement les protéines nouvellement synthétisées et mal repliées. On montre que, suite à leur modification par NEDD8/Ubiquitin, ces protéines sont transloquées du cytosol au noyau, où elles sont dégradées par le protéasome. Cependant, des conditions de stress prolongé causent une atténuation de l’activité nucléaire des protéasomes 26S, ce qui provoque alors l’accumulation des protéines endommagées sous forme d’inclusions nucléaires. Ces dernières sont réversibles et peuvent être éliminées par le protéasome une fois le stress atténué. Afin d’identifier les cibles de NEDD8 dans des conditions de stress, nous avons développé une approche protéomique basée sur une stratégie de mutation ponctuelle (NEDD8R74K). Cette stratégie permet l’identification des sites spécifiques de NEDDylation au sein des protéines cibles. Cette approche en combinaison avec le SILAC a permis l’identification de NEDD8, Ubiquitine, SUMO-2 et les protéines ribosomiques en tant que principales cibles de NEDD8 en réponse au stress. Ce qui était plus intéressant est que, en appliquant l’étude protéomique SILAC, on a pu constater que le rôle essentiel de la NEDDylation atypique est d’induire l’agrégation/séquestration d’un ensemble spécifique de protéines au sein des inclusions nucléaires. De plus, nous avons montré que l’agrégation induite par NEDD8 protège les protéasomes nucléaires d’une sévère déficience et permet une meilleure survie cellulaire pendant le stress. Notre étude présente NEDD8 comme un nouvel effecteur dans le réseau de protéostasie, elle identifie une nouvelle inclusion nucléaire cytoprotectrice et montre que la NEDDylation atypique est essentielle pour la réponse cellulaire au stress. / Cells are continuously endangered by a variety of proteotoxic stresses that cause protein misfolding and accumulation. Defects in repair or elimination of protein damage can lead to the formation of toxic aggregates that have been associated with diseases, such as neurodegenerative disorders and cancer. To prevent this toxicity, cells have evolved multiple quality control processes that interact and cooperate to maintain protein homeostasis leading to cellular fitness. These processes form “the proteostasis network”, and include molecular chaperones, proteolytic machineries (lysosomes, proteasomes) and pathways for protein damage sequestration. One of the main effectors of this network is the Ubiquitin and the Ubiquitin-like molecules, such as SUMO and NEDD8. These molecules covalently modify proteins through the action of E1, E2 and E3 enzymes. Historically, it was believed that each pathway employed its own and unique set of enzymes to post-translationally modify its substrates. Ubiquitination is essential for the cellular response to stress, especially by targeting misfolded proteins for proteasomal degradation. However, we recently discovered that proteotoxic stresses including proteasome inhibition, heat shock and oxidative stress induce a global increase in protein NEDDylation. Surprisingly, this increase does not depend on the NEDD8 activating enzyme NAE, but rather on the Ubiquitin activating enzyme Ube1, and is characterized by the formation of poly-NEDD8 chains and mixed chains between NEDD8 and Ubiquitin. Importantly, this process is reversible and cell recovery is accomplished once stress is alleviated. In this study, we focused on characterizing the NEDD8 response to stress or “atypical NEDDylation” in order to understand its biological relevance under these conditions.Our results showed that atypical NEDDylation depends on Hsp70/90 and targets mainly newly synthesized damaged proteins. We showed that, after their NEDDylation/Ubiquitination, misfolded proteins are progressively translocated from the cytosol into the nucleus for proteasomal degradation. However, upon prolonged stress conditions, the activity of nuclear 26S proteasome is compromised, resulting in the accumulation of these conjugates into nuclear inclusions. These inclusions are reversible and eliminated by nuclear proteasomes once stress is alleviated. In order to identify NEDD8 targets upon these conditions, we developed a proteomic approach based on a point mutation strategy (NEDD8R74K) that enables a site-specific analysis of NEDDylated proteins. This approach in combination with SILAC allowed the identification of NEDD8, Ubiquitin, SUMO-2, and ribosomal proteins as the major NEDD8 targets upon stress. Interestingly, by SILAC proteomics we found that the main function of atypical NEDDylation is to induce the aggregation/sequestration of a specific subset of proteins within the nuclear inclusions. We showed that this NEDD8-induced aggregation protects nuclear proteasomes from a severe impairment and allows a better cell survival upon proteotoxic stress.Our study defines NEDD8 as a new effector in the proteostasis network, identifies a new cytoprotective nuclear inclusion and shows that atypical NEDDylation is essential for the cellular response to stress.

Small Heat Shock Proteins (sHSP) are critical molecular chaperones that function to maintain protein homeostasis (proteostasis) and prevent the aggregation of other proteins during cellular stress. Any disruption in the process of proteostasis can lead to prevalent diseases ranging from cancer and cataract to cardiovascular and Alzheimer's disease. CryAB (αB-crystallin, HspB5) is one of ten known human sHSP that is abundant in the lens, skeletal, and cardiac muscle. This protein is required for cardiac function and muscle cell integrity. When the cell experiences physiological stress, including heat shock, CryAB moves to the cytoskeleton to act as a chaperone and prevent aggregation of its protein clientele. This research is designed to investigate the molecular role of CryAB in cell proteostasis through the identification of putative protein clientele and chaperone activity analysis. We have identified over twenty CryAB-binding partners through combined yeast two-hybrid (Y2H) and co-purification approaches, including interactions with myofibril proteins. Previously reported disease-associated CryAB missense variants were analyzed in comparison to wild type CryAB through Y2H binding assays. The characterization of the similarities and differences in binding specificities of these variants provide a foundation to better understand the chaperone pathways of CryAB and how these changes in molecular function result in the development of disparate diseases such as cataract, cancer, and various myopathies.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.

La maladie d'Alzheimer est une pathologie neurodégénérative progressive liée à l'âge qui détériore premièrement les fonctions liées aux mémoires de travail et épisodiques, avant de s'étendre à l'ensemble des procédures mémorielles dans les stades plus avancés. L'ensemble des traitements existant à ce jour sont palliatifs. Au niveau histologique, la maladie d'Alzheimer est caractérisée par l'accumulation extra- et intracellulaire de différentes protéines agrégées (appelées amyloïde) dans les tissus cérébraux, entrainant des dysfonctions importantes du circuit neuronal. De fait, la majorité des approches thérapeutiques en développement consistent à tenter de réduire ou supprimer ces agrégats protéiques. Cependant, la maladie d'Alzheimer étant étroitement corrélée au vieillissement, certaines de ses caractéristiques biologiques sont parfois confondues avec celles du vieillissement non pathologique. L'une de ces caractéristiques est la diminution des différents mécanismes liés à l'homéostasie protéique (protéostasie). L'hypothèse réalisée au cours de mes travaux est que le rétablissement de ces mécanismes diminués par l'âge constituerait une approche thérapeutique crédible, complémentaire aux approches actuelles, à la pathologie complexe qu'est la maladie d'Alzheimer. C'est en suivant cette optique que je me suis intéressé au rôle et à la régulation de l'un des systèmes majeusr du contrôle de la protéostasie : l'UPR (unfolded protein response), et en particulier au facteur de transcription XBP-1s, considéré comme l'une des pièces maîtresses de ce réseau de signalisation cellulaire / Alzheimer's disease is a neurodegenerative pathology strongly correlated to aging. Its symptoms are characterized by an impaired short term memory process in the early stages of the disease and later on by a loss of all type of memory process. There is actually no cure for this pathology. At the histo-pathological levels, the disease show an accumulation of aggregated proteins in the brain (called amyloid protein) in the intra or extra cellular space, which act as a disruptor of the normal neuronal function and activity. Thus, most of the therapeutic approach to treat the disease aim at removing those proteins aggregates from the brain. However, some of the Alzheimer's disease characteristics could be melded with normal aging : One such case is the global decrease of the proteostasis mechanism in the cell which normally happen in normal brain. The assumption made during this work is that the recovery of these mechanisms impaired by age would constitute a credible therapeutic approach, complementary to the other existing approaches to the complex disease that is Alzheimer's disease. Following this hypothesis I was interested in the role and regulation of one of the major system controlling proteostasis: the UPR (unfolded protein response), and particulary to the XBP-1s transcription factor , considered one of the master regulator of this cellular network