The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 Å from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions. Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.

The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)

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It is predicted that by 2050 there will be 10 million deaths annually due to super-resistant bacterial infections. Antimicrobial resistance (AMR) is already responsible for nearly 5 million deaths a year. Ribosomes serve as an ideal drug target being frequently targeted by antibiotics and having a highly conserved structure with few options for resistance. However, computer aided drug design (CADD) using ribosome crystal structures presents several challenges and is underutilized in the field. In this work we establish a successful protocol for antibiotic redocking and docking within the high interest sites of the peptidyl transferase center (PTC). Molecular visualization and interaction mapping were used to atomistically delineate binding patterns in the ribosomal PTC that could be used for CADD. Eleven ribosome crystal structures were validated for computational testing, which revealed derivative binding patterns in the A-site and P-site that can be used to increase antibiotic efficacy. Ribosome overlays revealed high interaction frequency nucleotides that were widely conserved throughout the different species and could be used to inform bidentate design to target two pockets at once. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR. / Master of Science in Life Sciences / Antimicrobial resistance (AMR) to antibiotics by bacteria is a rapidly increasing problem. Current trends predict that there will be more death due to super-resistant bacterial strains than cancer by 2050. Ribosomes are essential cellular machinery for bacteria and make an ideal antibiotic target. Using computational tools to optimize antibiotics with available ribosome crystal structural data presents several challenges and is underutilized throughout the field. In this work we establish a successful protocol for determining and exploiting antibiotic binding patterns within the functional center of the ribosome, the peptidyl transfer center (PTC). Nearly a dozen ribosome crystal structures were validated for computational testing, and binding patterns were revealed within the PTC that allowed antibiotic derivatives with increased efficacy to be developed. Ribosome validation also helped inform new drug class design so that multiple drug sites could be targeted at once, which were docked sharing high frequency nucleotide interactions with both parent antibiotics. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR.

Les maladies à prion font partie des maladies neurodégénératives. L’agent responsable est la protéine prion PrPSc. La conversion de la forme cellulaire nativePrPC en forme pathologique PrPSc et son agrégation sous forme des fibres amyloïdeconstituent des éléments clés de la physiopathologie des maladies à prion. Pourtant,les mécanismes contrôlant/favorisant cette conversion sont très mal connus. Chez lalevure Saccharomyces cerevisiae, il n’existe pas d’homologue de la protéine PrP,mais des protéines se comportant comme des prions existent, telle que Sup35p quiest responsable du prion [PSI+] ou encore la protéine Ure2p qui est responsable duprion [URE3]. Lors d’études antérieures à cette thèse, le laboratoire a isolé la 6AP etle GA, des molécules actives contre les prions de levure [PSI+] et [URE3] et contre leprion de mammifère PrPSc dans des tests cellulaires ainsi que in vivo dans unmodèle murin pour les maladies à prion. Ces résultats démontrent au moins certainsdes mécanismes de prionisation sont conservés de la levure aux mammifères.L’équipe a ensuite montré que la 6AP et le GA étaient des inhibiteurs spécifiques etcompétitifs de l’activité chaperon de protéines du ribosome (ou PFAR pour ProteinFolding Activity of the Ribosome). Ces résultats suggéraient donc que l’activité PFARreprésente un nouveau mécanisme de prionisation conservé de la levure auxmammifères. Par ailleurs, la 6AP et le GA s’étant révélées actives dans des modèlespour d’autres maladies neurodégénératives à fibres amyloïdes, l’activité PFARpourrait également être un acteur physiopathologique majeur de ces protéinopathies.Ma thèse avait deux objets : tester l’implication de l’activité PFAR dans l’apparitionet/ou la propagation des prions et enfin identifier de nouvelles molécules antiprion etcomprendre leurs mécanismes d’action. Mes résultats montrent que l’activité PFARjoue bien un rôle dans la propagation des prions de levure. En effet, l’enrichissementen PFAR favorise l’apparition spontanée du prion [PSI+]. Il conduit également à uneinstabilité accrue de ce même prion. Ainsi, l’activité PFAR ressemble à celle duchaperon de protéine Hsp104p, une protéine indispensable au maintien et à lapropagation de tous les prions de levure, mais qui n’a pas d’homologue chez lesmammifères. Mes résultats suggèrent que les activités PFAR et Hsp104p sontpartiellement redondantes pour le maintien des prions chez la levure et que, chez lesmammifères, seule l’activité PFAR jouerait ce rôle. Parallèlement, nous avonsidentifié de nouvelles familles de molécules antiprion, actives tant contre les prionsde levure que de mammifères. Ces molécules inhibent toutes l’activité PFAR. Nosrésultats contribuent ainsi à une meilleure compréhension des mécanismes deprionisation. Ils indiquent également que l’activité PFAR est une cible thérapeutiqueprometteuse pour les maladies à prion, mais aussi probablement pour d’autresprotéinopathies beaucoup plus fréquentes. / Prion diseases are considered neurodegenerative diseases. The incriminated agentis the prion protein PrPSc. The conversion of PrP from its native conformation PrPC tothe pathologic form PrPSc is the major element of the pathogenesis of prion diseases.However, the mechanisms involved in this conversion are poorly understood. In theyeast Saccharomyces cerevisiae, there is no counterpart of the PrP protein. Howeverproteins acting as prion do exist in yeast, such as the Sup35 protein responsible forthe prion [PSI+], or the Ure2 protein responsible for the prion [URE3]. In previousstudies, our team isolated two compounds, 6AP and GA, which are active against theyeast prions [PSI+] and [URE3 ] and against the mammalian prion PrPSc in cellbasedassays as well as in vivo in a mouse model for prion diseases. These resultsdemonstrated that the prionisation mechanisms are at least partially conserved fromyeast to mammals. 6AP and GA specific and competitive inhibitors of the ProteinFolding Activity of the Ribosome (PFAR) thereby showing that the PFAR is oneconserved mechanism of the prionisation. Moreover, 6AP and GA have been provenactive against other amyloid diseases thus placing the PFAR as a key player in thepathophysiology of protein folding diseases. My thesis aims were to test theinvolvement of the PFAR in the initiation and / or propagation of prion, to identify newantiprion molecules and to understand their mechanisms of action. My results showthat the PFAR plays a central role in the yeast prion propagation. Indeed, PFARenrichment promotes the spontaneous appearance of the prion [PSI+] and at thesame time leads to an increased instability of the same prion. Thus, PFAR activityresembles the yeast Hsp104p chaperone protein activity in the maintenance andpropagation of all yeast prions. My results suggest that the PFAR and Hsp104pactivity are partially redundant and that only the PFAR should play this role inmammals. Meanwhile, we have identified new antiprion drugs that are active againstboth yeast and mammal’s prions. These compounds are all inhibitors of the PFAR.Our results contribute to a better understanding of the prionisation mechanisms andindicate that the PFAR is a promising therapeutic target for prion diseases andprobably also for common protein folding diseases.Keywords: prion, yeast, ribosome, protein chaperon, Hsp104

L’hématopoïèse est un processus complexe et extrêmement régulé qui permet la production de l’ensemble des cellules sanguines à partir de cellules souches. Différents acteurs interviennent dans cette régulation et une altération de l’un ou plusieurs de ces régulateurs est souvent à l’origine de leucémies. L’un des acteurs majeurs de cette régulation est le complexe Core Binding Factor (CBF), particulièrement touché dans ces hémopathies. Ce facteur de transcription se compose de la sous-unité CBFβ et d’une sous unité variable RUNX, (habituellement RUNX1 dans l’hématopoïèse). Dans la leucémie aiguë myéloïde 4 à composante éosinophile (LAM4 Eo), le gène CBFβ est retrouvé fusionné au gène MYH11, entraînant la formation d’un gène chimérique codant pour l’oncoprotéine de fusion CBFβ–SMMHC. Cette version altérée du complexe CBF a pour caractéristique de séquestrer RUNX1 dans le cytoplasme et de déréguler l’expression des gènes cible du complexe via diverses mécanismes. Elle est en effet capable d’inhiber l’expression génique par le recrutement d’inhibiteurs transcriptionnels mais a également récemment été décrite comme liée au promoteur de gènes actifs. Ces dérégulations entraînent une altération de la différenciation et/ou une apoptose chez différents progéniteurs hématopoïétiques via divers mécanismes particulièrement étudiés chez la souris. Chez l’homme, les processus oncogéniques par lesquels CBFβ–SMMHC altère la différenciation et induit la leucémogénèse restent cependant peu décrits. Au moyen de deux modèles humains : une lignée ME-1 inductible pour l’inhibition de l’expression de l’oncoprotéine et des blastes leucémiques de patients atteints de LAM4 Eo dérivés de xénogreffes murines, nous avons découvert un nouveau composant cellulaire dérégulé par CBFβ–SMMHC ainsi qu’un nouveau partenaire d’interaction. En effet, dans un premier temps, ce travail révèle que l’oncoprotéine a des effets complexes sur la biogenèse des ribosomes aux niveaux génomique et post-transcriptionnel. Nous avons montré que CBFβ–SMMHC fixe le promoteur des gènes ribosomiques et active leur transcription. Nous avons également observé un niveau d’expression de ces gènes, supérieur dans les LAM dites de type CBFβ–SMMHC comparées aux autres sous-groupes de LAM. Dans la lignée ME-1 cette activation de la transcription ne se traduit cependant pas par une augmentation du contenu cellulaire en ribosomes, expliqué en partie par une maturation du précurseur des ARN ribosomiques moins efficiente en présence de l’oncoprotéine. Dans un second temps nous avons observé que CBFβ–SMMHC interagit directement avec la protéine Polycomb RING1B et BMI1 sous-unité du complexe de répression des gènes PRC1. L’inhibition de CBFβ–SMMHC entraînant une augmentation du niveau de fixation globale de RING1B sur l’ensemble du génome. Nous pensons que de cette altération du niveau de fixation de RING1B induite par CBFβ–SMMHC, découle la dérégulation de nombreux gènes impliqués dans diverses voies ou mécanismes critiques de l’hématopoïèse. Nous avons ainsi mis en lumière deux nouveaux mécanismes oncogéniques médiés par l’oncoprotéine CBFβ–SMMHC ouvrant de nouveaux horizons pour de potentielles cibles thérapeutiques. / Haematopoiesis is a complex process allowing the production of all mature blood cells from stem cells. This process is highly regulated at the transcriptional level, and perturbation of normal transcriptional regulation may cause leukaemia. One of the major actors of this regulation is the Core Binding factor (CBF) complex, which is frequently subject to genetic alteration in leukaemia. This transcription factor consists of a constant CBFβ subunit and a variable RUNX subunit, usually RUNX1 in haematopoiesis. In acute myeloid leukemia 4 with eosinophilic component (AMLM4 Eo), the CBFβ gene is fused to the MYH11 gene, leading to the formation of a chimeric gene encoding the CBFβ–SMMHC oncoprotein. This altered version of the CBF complex sequesters RUNX1 into the cytoplasm, and deregulates wild type CBF target gene expression though diverse mechanisms. While CBFβ–SMMHC can inhibit gene expression by recruiting transcriptional inhibitors, it has also recently been described to bind and activate certain gene promoters. The mechanisms by which these deregulations lead to an alteration of the differentiation and/or an apoptosis of diverse hematopoietic progenitors is best characterised in murine models. In humans, the oncogenic processes by which CBFβ–SMMHC alters differentiation and induces leukaemogenesis remain unclear. Using two human cellular models, namely (i) an ME-1 cell line containing an inducible shRNA directed against the CBFβ- MYH11 fusion transcript and (ii) Patient-derived AML M4Eo murine xenografts, we describe two novel activities of CBFβ–SMMHC. Firstly, we discovered that the oncoprotein has complex effects on ribosome biogenesis at both the genomic and post-transcriptomic levels. We found that CBFβ–SMMHC binds ribosomal gene promoters and activates their transcription, which was corroborated by the observation of higher ribosomal gene expression in human AML M4Eo, compared with other AML subgroups. In the ME-1 cell line this transcriptional activation did not lead to the higher cellular ribosome content, which was explained in part by decreased efficiency of ribosomal RNA maturation in the presence of the oncoprotein. Secondly, we found that CBFβ–SMMHC interacts directly with RING1B and BMI1 protein subunit of the Polycomb gene repression complex PRC1. Depletion of CBFβ–SMMHC lead to increased global binding of RING1B to the genome, resulting in deregulation of numerous genes that are critical for normal haematopoietic differentiation. We have therefore highlighted two new oncogenic mechanisms mediated by the CBFβ–SMMHC oncoprotein, therefore opening new avenues to investigate potential therapeutic targets.

Le ribosome est un organite essentiel de la cellule qui assure la synthèse des protéines. C'est une structure très conservée, composée d'ARN et de protéines ribosomiques organisés en deux sous-unités. Les expériences de reconstitution in vitro du ribosome d'E. coli ont montré que l'assemblage est un processus coordonné impliquant de nombreuses interactions entre les différents constituants. En particulier, les premières étapes de l'assemblage de la grande sous-unité dépendent fortement de la fixation coopérative de cinq protéines ribosomiques à l'ARN 23S, mais les mécanismes moléculaires sous-jacents sont mal connus.Cette étude à l'échelle de la molécule unique vise à préciser ces mécanismes et porte sur un fragment constitué des hélices H18, H19 et H20 du domaine I de l'ARN ribosomique 23S contenant les sites de fixation des protéines uL24 et uL4. Ce fragment d'ARN a été préparé dans une configuration qui permet la mesure de force via un double piège optique. Les courbes de force obtenues ont permis de dresser une cartographie de la stabilité des structures du fragment d'ARN.Ces cartes ont été comparées en absence et en présence des protéines ribosomiques uL24 et/ou uL4, démontrant ainsi que le fragment d'ARN est stabilisé par la fixation des protéines uL24 et/ou uL4. Leur fixation est coopérative et la présence conjointe des deux protéines sur-stabilise les structures du fragment d'ARN.Ces résultats sont discutés dans la perspective de préciser le rôle du fragment d'ARN et des protéines ribosomiques uL24 et uL4 dans l'assemblage de la grande sous-unité du ribosome. / Ribosomes are essential organelles of the cell responsible for the synthesis of proteins. Their well conserved structure made of RNA and proteins is organized into two subunits. In vitro reconstitution of E. coli ribosomes showed that their assembly is a coordinated process which involves many interactions between the components. More specifically, the early stages of the large subunit assembly depend strongly on the cooperative binding of five ribosomal proteins to the 23S RNA. The underlying molecular mechanisms however remain poorly understood.The aim of this study is to shine new light on these mechanisms at the single molecule level. It focuses on a 23S ribosomal RNA fragment composed of the helices H18, H19 and H20 in domain I which encompasses the binding sites of the ribosomal proteins uL24 and uL4. This RNA fragment has been prepared in a dumbbell configuration and force versus displacement measurements have been performed using a dual optical trap. From these measurements, a map summarizing the mechanical stability of the RNA fragment has been determined.The maps obtained in absence and in presence of the ribosomal proteins uL24 and/or uL4 have been compared consequently demonstrating mechanical stabilization of the RNA fragment induced by the binding of uL24 and/or uL4. Moreover, their binding is cooperative and when both are present, the mechanical stabilization of the RNA fragment is enhanced.These results are discussed to specify the role of the RNA fragment and proteins uL24 and uL4 in the large ribosomal subunit assembly.

Le ribosome est un complexe macromoléculaire impliqué dans la synthèse protéique de toutes les cellules vivantes. L’étape d’élongation de cette synthèse est un processus itératif débutant par la sélection au sein du ribosome d’un ARNt aminoacylé suivie par le transfert du peptide du site P- vers le site A- et de la translocation de l’ARNm et de l’ARNt. Le facteur d’élongation 2 (eEF2), qui catalyse la translocation, est l’un des acteurs majeur de cette étape d’élongation chez les eucaryotes. Cependant le mécanisme par lequel eEF2 induit ce processus est encore aujourd’hui inconnu. Dans cette étude structurale, nous présentons la première structure à haute résolution (3.1 Å) du complexe de pré-translocation résolu par cristallographie aux rayons X. La structure obtenue nous a permis d’identifier les différents composants du complexe de translocation et de proposer le rôle de l’His699 et celui de la diphtamide, modification post-traductionnelle d’eEF2, lors du stade de pré-translocation. / Elongation is the longest stage of protein synthesis that takes place on the ribosome and represents a cycle that begins with an aminoacyl-tRNA selection followed by the catalysis of peptide transfer from the P- to the A-site and mRNA-tRNA translocation. Elongation factor 2 (eEF2) is one of the key player of elongation cycle in eukaryotes that catalyzes translocation of mRNA and tRNA on the ribosome. However the mechanism how eEF2 induces translocation on the ribosome is unknown. Current work investigates the structural aspect of protein synthesis machinery in eukaryotes. In particular we present first high resolution structure of functional pretranslocation complex solved at 3.1 A by X-ray crystallography. The obtained structure allowed us to see several features of translocation complex and to propose the role of His699 and post translational modification of eEF2 diphthamide during at pretranslocation stage.