This thesis is an investigation of novel mono-ADP-ribosylating toxins. In the current data-rich era, making the leap from sequence data to knowledge is a task that requires an elegant bioinformatics toolset to pinpoint questions. A strategy to expand important protein-family knowledge is required, particularly in cases in which primary sequence identity is low but structural conservation is high. For example, the mono-ADP-ribosylating toxins fit these criteria and several approaches have been used to accelerate the discovery of new family members. A newly developed tactic for detecting remote members of this family -- in which fold recognition dominates -- reduces reliance on sequence similarity and advances us toward a true structure-based protein-family expansion methodology. Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins identified and characterized using in silico and cell-based techniques. Medically relevant toxins from Mycobacterium avium and Enterococcus faecalis were also uncovered. Agriculturally relevant toxins were found in Photorhabdus luminescens and Vibrio splendidus. Computer software was used to build models and analyze each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. Yeast-based activity tests have since confirmed activity. Vibrio cholerae produces cholix – a potent protein toxin of particular interest that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 2.1Å apo X-ray structure as well as a 1.8Å X-ray structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD+). Hallmark catalytic residues were substituted and analyzed both for NAD+ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These new toxins serve as a reference for ongoing inhibitor development for this important class of virulence factors. In addition to using toxins as targets for antivirulence compounds, they can be used to make vaccines and new cancer therapies. / Natural Sciences and Engineering Research Council (CGS-D), Canadian Institutes of Health Research, Cystic Fibrosis Canada, Human Frontier Science Program, Ontario government (OGSST), University of Guelph (Graduate Research Scholarship)

Nicotinamide adenine dinucleotide (NAD+) represents a long-known key molecule in cellular metabolism. It was initially identified for its ability to convey electrons and protons between redox partners in multiple bioenergetic and biosynthetic reactions. In addition, NAD+ also serves as a substrate for NAD+-consuming enzymes such as sirtuins and poly ADP-ribose polymerases (PARPs). These latter enzymes catalyze dynamic post-translational modifications that control virtually every signaling pathway orchestrating cell fate. The aim of this work was to analyze the role of NAD+ in the context of programmed cell death mechanisms.<p>Our findings indicate that NAD+ is protective against DNA damage-induced cell death and FAS-induced apoptosis, while, unexpectedly, it promotes TNF-induced necroptosis, a regulated form of necrosis. Indeed raising NAD+ cellular levels sensitized culture cells to necroptosis, while NAD+ depletion protected cells from this form of cell death. Furthermore, specific silencing of NAD+-dependent sirtuins was also found to be protective against TNF-induced necroptosis. Consistently, a pharmacological pan-sirtuin inhibitor called cambinol protected cells from necroptosis. Then, as necroptosis represents a back-up mechanism that may have evolved in response to viral pathogens expressing anti-apoptotic proteins, we demonstrated in an in vitro model mimicking viral infection that pharmacological sirtuin inhibition protected cells from poly I:C-induced necroptotic cell death. In vivo, we demonstrated that cambinol partially protected kidney from necrosis after ischemia/reperfusion. We have also shown that enhancing liver NAD+ concentration via isonicotinamide increases the susceptibility of mice to systemic inflammatory response syndrome (SIRS). Moreover, our preliminary data show that isonicotinamide substantially improves the ability of cyclophosphamide to trigger the rejection of the murine mastocytoma P815 tumor cell line.<p>Collectively, our observations point to a role for NAD+ in the control of necroptosis in a sirtuin-dependent manner. These observations may bear relevance to the better understanding of the pathophysiological consequences of excessive production of the pro-inflammatory cytokine TNF and the control of viral infections and tumor progression/immunotherapy. & / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Poly(ADP-ribose) polymerases belong to a family of proteins with 17 members in human beings. PARP1, the founding member of the family is a protein that synthesizes linear or branched polymers of ADP-ribose on itself or on target proteins. Different members of this family, that do not all possess ADP-ribosyl polymerase activity, are involved in the regulation of various cellular mechanisms. Some members of the family are particularly involved in the positive or negative control of the immune response. PARP1 is a key player in the regulation of inflammation, through its positive control of cell death and of proinflammatory cytokine production. On the other hand, the tankyrases (PARP5a and PARP5b) and PARP14 seem to regulate inflammatory responses in a negative fashion. PARP12 is a poorly characterized member of the family, whose expression is greatly increase following stimulation with type-I interferons, cytokines mainly involved in antiviral defences.<p>PARP12 is a protein that possesses three main domains: A putative RNA binding N-terminal domain composed of tandem CCCH zinc-fingers, a central WWE domain and a C-terminal PARP catalytic domain. In this work, we have shown that the expression of PARP12 is strictly-dependent on type-I interferons, that it possesses ADP-ribosyl transferase activity and that in can regulate the translation of messenger RNA into proteins. PARP12 can be found in stress granules, sites of storage of untranslated mRNAs, and is capable of directly inhibiting the translation of a reporter mRNA when tethered to it, in a manner dependent on its catalytic activity. Furthermore overexpression of wild-type PARP12, in contrast to overexpression of a mutant with no detectable catalytic activity (PARP12-G575W), leads to a general arrest of most cellular translation.<p>On the other hand, we have shown that PARP12 can activate the transcription of genes under the control of an NFκB-dependent promoter, especially when its zinc-fingers are deleted or mutated (PARP12ΔZnF). PARP12ΔZnF is located in structures that can enclose TRIF, RIP1, NEMO, p62/SQSTM1 and ubiquitin. These proteins have all possess an important role in the activation of NFκB signalling cascades. Moreover, we have shown that endogenous PARP12 is situated in ALIS (Aggresome-Like Induced Structures) in LPS-stimulated macrophages. These structures have a possible role in the presentation of antigens on class I major histocompatibility complexes, implying that PARP12 may be involved in the regulation of antigen presentation. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L’ADN contient l’information génétique essentielle au développement et au bon fonctionnement de tout organisme vivant. Cependant, l’ADN peut être endommagé ou modifié par une exposition régulière à différents facteurs tels que la lumière du soleil, la pollution, la radiation, etc. La cellule a ainsi développé des mécanismes de réparation très efficaces puisqu’un ADN sain est essentiel pour la santé d’un organisme. La protéine humaine PARP1 est une enzyme clé de la réparation de l’ADN. PARP1 détecte rapidement les dommages à l’ADN en s’y liant, ce qui stimule son activité catalytique. PARP1 catalyse la formation de chaînes d’ADP-ribose qui sont ajoutées à PARP1, ainsi que d’autres protéines, en tant que modification post-traductionnelle. Les chaînes d’ADP- ribose permettent la décondensation de la chromatine ainsi que le recrutement de facteurs de réparation à l’ADN endommagé. PARP1 possède plusieurs domaines régulateurs en plus de son domaine catalytique, le domaine catalytique lui-même se divisant en un domaine hélicoïdal (HD) ainsi qu’un domaine ADP- ribosyltransférase. L’augmentation de l’activité catalytique de PARP1, à la suite de sa liaison à l’ADN endommagé, implique qu’un signal allostérique se transmette à travers ses différents domaines. Le HD joue un rôle essentiel dans le relais de cette communication allostérique puisque c’est ultimement un changement de conformation du HD (i.e « ouverture ») qui révèle le site actif et active l’enzyme. De plus, il a été démontré que les inhibiteurs de PARP1 peuvent moduler l’affinité de l’enzyme pour l’ADN endommagé. Certains inhibiteurs peuvent ainsi provoquer la « capture » de l’ADN par PARP1, un phénomène qui requiert la présence du HD et qui est particulièrement toxique pour les cellules cancéreuses présentant des défauts de réparation de l’ADN. Pour cette raison, la mort cellulaire induite par les inhibiteurs de PARP1 est un traitement prometteur et quatre inhibiteurs sont déjà utilisés pour traiter le cancer des ovaires et du sein. Cependant, le mécanisme précis derrière la capture de l’ADN par PARP1 est encore nébuleux et nécessite de plus amples recherches. Puisque la capture de l’ADN par PARP1 requiert le relais d’un signal allostérique par le HD, et que l’ouverture du HD participe à cette communication, il est donc essentiel de comprendre les changements de conformations effectués par ce domaine. Nous avons ainsi obtenu pour la première fois une structure atomique de PARP1 en conformation active. Celle-ci montre que l’ouverture du HD amène la formation d’une interface additionnelle entre ce domaine et les autres domaines régulateurs de PARP1. Ainsi, entraver la formation de cette nouvelle interaction, par des mutations ponctuelles, diminue grandement l’activité catalytique de PARP1 lié à l’ADN, ce qui suggère que l’interface participe à la communication allostérique de l’enzyme. Tel que mentionné plus haut, les inhibiteurs de PARP1 peuvent moduler de manières différentes l’affinité de PARP1 pour les dommages à l’ADN et ainsi influencer distinctement la communication allostérique de l’enzyme. Nous avons caractérisé une nouvelle série d’inhibiteurs de PARP1 et évalué leur capacité à moduler l’affinité de PARP1 pour l’ADN endommagé. Nos travaux démontrent qu’un inhibiteur volumineux occupant le site actif n’augmentera pas nécessairement l’affinité de PARP1 pour les dommages à l’ADN. Leur capacité à favoriser la capture de l’ADN dépend plutôt de leur interaction avec la région du HD voisine au site actif. En résumé, nos travaux participent à l’amélioration des connaissances concernant l’activation catalytique de PARP1 et la communication allostérique. Une meilleure compréhension de l’allostérie de PARP1 permettra la conception de médicaments ayant la toxicité désirée pour tuer les cellules cancéreuses. / DNA contains the genetic instructions for the development and proper function of all living organisms. However, DNA can be broken and modified in harmful ways through daily exposures to exterior stresses such as sun light, pollution, radiation, etc. Since stable and undamaged DNA is essential for the health of an organism, cells have developed repair mechanisms to ensure that DNA damage is taken care of efficiently. The human protein PARP1 is a key enzyme that contributes to DNA repair. PARP1 rapidly detects DNA lesions which greatly stimulates its catalytic activity. PARP1 catalyzes the formation of chains of ADP-ribose that are attached covalently to PARP1 itself, or other target proteins, as a posttranslational modification. The chains of ADP-ribose allow for the recruitment of chromatin remodelling factors and repair factors to process the DNA lesions. PARP1 carries multiple regulatory domains in addition to its catalytic domain, with the catalytic domain itself composed of the helical domain (HD) and the ADP-ribosyltransferase fold. DNA damage binding greatly stimulates PARP1 catalytic activity, which requires that an allosteric signal is relayed across the enzyme’s domains. Interestingly, the HD has been found to play an essential role in the PARP1 allostery. The HD undergoes a change of conformation (i.e. opening) following PARP1 DNA damage binding which reveals the active site. Additionally, inhibitors binding the active site of the enzyme can modulate PARP1 DNA binding affinity. Some inhibitors can induce PARP1 DNA “trapping”, a phenomenon that requires the HD and appears particularly toxic to cancer cells bearing DNA repair deficiencies. Cell death induced by PARP1 inhibitors is a promising cancer treatment and four inhibitors have approval for clinical use against ovarian and breast cancers. However, the precise mechanism underlying PARP1 trapping on DNA is still unclear and requires further research. Since PARP1 trapping requires the presence of the HD, and that the HD opening is involved in relaying the allosteric signal, it remains essential to characterize its change of conformation. We have obtained for the first time atomic structures of PARP1 in a catalytically active state. Crystal structures show that the HD in open conformation forms an additional interdomain interface. Mutating this interface prevents PARP1 strong catalytic activation following DNA damage binding, suggesting that the allosteric communication is impaired. Additionally, these structures reveal how the HD active conformation leads to the reveal of the active site in the ART domain. As mentioned above, PARP1 inhibitors can modulate the enzyme’s DNA binding affinity and therefore impact its allosteric communication. We have characterized a series of novel inhibitors and tested their propensity to increase PARP1 DNA binding affinity. Our work highlights that bulky inhibitors that fill the active site will not necessarily promote PARP1 affinity for DNA lesions. Rather, it appears that inhibitors may trigger DNA trapping via their interaction with a neighboring region of the HD. Overall, our work deepens our understanding of PARP1 catalytic activation and allosteric communication. Properly understanding how PARP1 trapping occurs will help the design of specific drugs with the desired toxicity to kill cancer cells.