The NF kappa B family of heterodimeric transcription factors is activated by many stimuli, and lead to the upregulation of countless genes. Not surprisingly, NF kappa B plays a critical role in many aspects of cellular function. In T and B lymphocytes, antigen receptor stimulation leads to the activation of NF kappa B through a signal transduction cascade involving the Bcl10-MALT1 complex. We hypothesized that this complex may be critical to signalling cascades other than those emanating from antigen receptors. B cell activation factor of the TNF family (BAFF) activates non-canonical NF kappa B heterodimers that promote B cell survival. Here, we show that MALT1 is required for BAFF-induced phosphorylation of NF kappa B2 (p100), p100 degradation and RelB nuclear translocation in B220+ B cells. TRAF3, a known negative regulator of BAFF-R mediated signaling, interacts with MALT1 in a manner which is negatively regulated by BAFF, and TRAF3 levels are enhanced in MALT1-/- B cells. MALT1-/- CD21highCD23low (MZ) B cells show a defect in BAFF-induced survival and MALT1-/- x BAFF-transgenic (Tg) mice have decreased MZ and B1 B cell levels compared to BAFF-Tg mice. In agreement with this in vitro data, phenotypes associated with over-expression of BAFF including increased serum immunoglobulin titres, spontaneous germinal center (GC) formation, and immune complex deposition in the kidney were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction. The mechanism by which the Bcl10-MALT1 complex regulates antigen induced NF kappa B activation in T cells remains controversial. To shed light on this regulatory network, we conducted biochemical purification of Bcl10, and identified Uev1a, a known regulator of antigen receptor mediated NF kappa B activation. We hypothesized that mms2, and structurally similar molecule to Uev1a, may also impinge on NF kappa B activation. Mms2 overexpression in 293T cells inhibited the Bcl10-induced activation of an NF kappa B sensitive luciferase. Lymphocyte development and antigen receptor induced activation occurs normally mms2-/- mice. However, class switched serum immunoglobulins, and survival responses to DNA damage inducing gamma-irradiation, are decreased in mms2-/- mice. Therefore, mms2 is dispensible in vivo for lymphocyte function and development, but is required for DNA damage responses.

L’ubiquitination est une modification posttraductionnelle (MPT) qui gouverne la plupart des processus cellulaires Eucaryotes. Elle consiste en l’ajout d’une ubiquitine via une liaison covalente à une protéine cible, altérant son devenir (activité, localisation, dégradation). Le LUBAC (linear ubiquitin chain assembly complex) est le seul complexe capable de catalyser une forme énigmatique de cette modification, l’ubiquitination linéaire. Ces dernières années, ce complexe ternaire a été identifié comme un acteur majeur dans l’activation de NF-κB en réponse à de nombreux immunorécepteurs. Le but principal de cette thèse est de caractériser la régulation du LUBAC et ses partenaires dans des systèmes variés et d’identifier d’éventuelles MPT en charge de moduler ce complexe. Dans un premier temps, nous avons montré que la sous-unité du LUBAC HOIL1 est dynamiquement clivée par la paracaspase MALT1 en réponse à l’engagement des récepteurs antigéniques dans les lymphocytes pour pleinement activer NF-κB. HOIL1 est également constitutivement clivée dans un sous-groupe agressif de lymphome diffus à grandes cellules B (DLBCL), qui présentent une activation aberrante de MALT1, dévoilant de potentielles stratégies thérapeutiques. Dans un second temps, nous avons observé qu’une partie du partenaire du LUBAC CYLD est liée aux satellites centriolaires, des structures granulaires qui gravitent autour des centrosomes et orchestrent la ciliogenèse. Nos données suggèrent que CYLD contrôle la protéostase des satellites centriolaires et gouverne ainsi la formation du cil primaire. Enfin, nous avons analysé les phases précoces de la nécroptose, une forme de nécrose sous le contrôle du LUBAC. Nous avons mis en évidence que l’activation des canaux Pannexin-1 restreint la production de cytokines pro-inflammatoires associée à la nécroptose. Ensemble, ces travaux illustrent comment les MPT contrôlent les voies de signalisation cellulaires associées à l’ubiquitine et comment elles peuvent être corrompues dans des conditions pathologiques. / Ubiquitination is a pivotal multifaceted post-translational modification (PTM), which governs most cellular processes in Eukaryotic cells. Ubiquitination consists in the covalent binding of ubiquitin onto a target protein, altering its fate (activity, localisation, degradation). The “linear ubiquitin chain assembly complex” (LUBAC) is the only known complex capable of catalysing the enigmatic “head-to-tail” linear ubiquitination. Over the years, this tertiary complex has been shown to be essential for signalling to the NF-κB transcription factors family in response to the stimulation of various immunoreceptors. The primary goal of this PhD was to better understand how the LUBAC and its partners are modulated in different systems and identify possible PTMs regulating this complex. First, we discovered that the LUBAC subunit HOIL1 is dynamically cleaved by the paracaspase MALT1 upon antigen receptor engagement in lymphocytes to further NF- κB activation. HOIL1 is also constitutively cleaved in a subset of aggressive diffuse large B-cell lymphoma (DLBCL), which displays aberrant activation of MALT1, unveiling potential therapeutic strategies. Second, we observed that a portion of the LUBAC’s partner CYLD is bound to the centriolar satellites, a set of granular structures surrounding centrosomes that orchestrate ciliogenesis. Our data suggests that CYLD governs the proteostasis of centriolar satellites, and thereby the formation of primary cilia. Lastly, we analysed the initial steps of necroptosis, a regulated form of necrosis under the control of the LUBAC. We found that activation of Pannexin-1 channels restrains the production of proinflammatory cytokines associated with necroptosis. Altogether, this work illustrates how PTM finely tune ubiquitin-associated signalling pathways, and how they can be perverted in pathological conditions.

Overexpression of the Receptor for Advanced Glycation End Products (RAGE) has been implicated in multiple diseases, including several types of cancer. In different types of cancer, RAGE has been shown to promote cell survival by either autophagy or activation of the transcription factor NF-κB. Based on what is known about RAGE, we hypothesized that the RAGE/ligand interaction at the cell surface promotes pancreatic cancer and melanoma cell survival by both pathways, autophagy and NF-κB activation. To study the role of RAGE in pancreatic cancer resistance to chemotherapy, BxPC-3, MIA PaCa-2, PANC-1, and RAGE overexpressing PANC-1 FLR2 cell-lines were used. A significant decrease in cell viability was observed upon gemcitabine treatment with further significant reduction in cell viability upon combination of gemcitabine with the RAGE inhibitor IgG 2A11. In our studies we showed that RAGE plays a central role in pancreatic cancer cell resistance to gemcitabine by increasing autophagy. To test the importance of RAGE localization in mediating drug resistance, three melanoma cell-lines (WM115, WM266, and SK-MEL2) with their daughters, RAGE overexpressing cells (WM115-RAGE, WM266-RAGE, and SK-MEL2-RAGE) were used. Wild type cell-lines only expressed RAGE intracellularly while RAGE overexpressing cells expressed RAGE both at the cell surface and inside cells. We show in this study that only the cell surface RAGE is involved in melanoma resistance to dacarbazine. We next tested the effects of RAGE/RAGE ligand interaction at the cell surface in pancreatic tumor growth. We used two carcinoma cell-lines, PANC-1 and MIA PaCa-2, for this purpose. Both cell-lines were transiently transfected with a NF-κB/Luciferase reporter plasmid to test the effects of the interaction between RAGE and its ligands on the activation of the NF-κB signaling pathway. We observed higher NF-κB activity upon treatment with RAGE ligands (AGE, S100P, and S100A8/A9) compared to non-treated cells. Higher activity of NF-κB was coupled with a higher expression of cyclin D1 and lower expression of p53, NF-κB target genes. / Cobre grant "P20GM109024"

Sodium methyldithiocarbamate (SMD) is commonly reported to cause health risks in humans. Previous reports indicate SMD causes oxidative stress, which can contribute to the activation of NF-êB and cause other characteristics of inflammatory responses to be altered. Almost all pro-inflammatory cytokines require NF-êB activation for full expression and development of an innate immune or inflammatory response. This study evaluated NF-êB activation, providing new information regarding reactive oxygen in macrophages from SMD-treated mice. Studies were conducted in which NF-êB reporter mice were treated with lipopolysaccharide (LPS), SMD, buthionine sulfoximine (BSO), and N-acetyl cysteine (NAC). BSO depletes glutathione (GSH) and increases oxidative stress, whereas NAC spares GSH by acting as a precursor for rapid synthesis to replace oxidized GSH. The work here indicates that NF-êB is not affected directly by increased or decreased reactive oxygen species (ROS), and oxidative stress is not the major mechanism by which SMD inhibits inflammatory responses.

L’infection par le VIH-1 est un problème majeur de la santé publique qui touche plus de 35 millions de personnes à l’échelle mondiale. La réplication du VIH-1 est déclenchée par l’activation du promoteur LTR, qui contient deux sites de liaison pour le facteur de transcription NF-κB. Ces sites de liaison sont hautement conservés dans le génome du VIH-1, illustrant ainsi l’importance de NF-κB dans l’activité transcriptionelle des gènes du VIH-1 et la production de nouvelles particules virales. La withaferin A (WA) est une substance bioactive extraite de la plante Withania somnifera, qui possède des propriétés pharmacologiques non négligeables dans la régulation de la réponse immunitaire. Des études récentes ont démontré que le potentiel anti-inflammatoire de la WA est dû principalement à l’inhibition de la voie de NF-κB. Le but de ce projet est de déterminer l’effet de la WA sur la réplication du VIH-1 dans les cellules T, qui sont les cibles principales du virus. Des essais de transfections transitoires de cellules T Jurkat E6.1 avec des plasmides contenant le promoteur du VIH-1 ayant différentes constructions de NF-κB, ont démontré que la WA peut réduire l’activité du promoteur d’une manière dépendante de NF-κB. Quant à la production de particules virales, des essais d’infection avec des virus pseudotypés démontrent que la WA diminue la production virale jusqu’à 90% dans des cellules T stimulées avec PMA/PHA et TNF-α, tandis que les mutants ayant des sites de liaison défective pour NF-κB ne sont pas affectés. Des essais de retardement sur gel ainsi que des immunobuvardages de type Western ont montré que la WA altère l’habilité de NF-κB à transloquer dans le noyau, ce qui se traduit par l’inhibition de la synthèse de l’IκB-α, protéine inhibitrice de NF-κB, phénomène sous contrôle étroite de ce facteur de transcription. Ces résultats suggèrent que la WA pourrait permettre une diminution de la réplication virale d’une manière dépendante de NF-κB et ainsi empêcher la propagation du virus aux cellules T chez les individus infectés.

No description available.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

Le but de ce travail était d'étudier l'activité anti-leucémique de UNBS1450, un cardénolide semi-synthétique appartenant à la famille des glycosides stéroïdes cardiaques, et qui a été démontré d'être doué d'un puissant potentiel inducteur d'autophagie dans les cellules cancéreuses issues de tumeurs solides. Par cette étude, nous démontrons pour la première fois que des concentrations nanomolaires de UNBS1450 exercent un effet pro-apoptotique sur des cellules leucémiques humaines. Par conséquent, nous avons élucidé les mécanismes moléculaires impliqués : nos résultats mettent en évidence une inhibition de la transactivation de NF-kB et une induction de l'apoptose par clivages des pro-caspases 8,9 et 3/7, en réprimant l'expression de Mcl-1 anti-apoptotique et en recrutant Bak et Bax, deux protéines pro-apoptotiques, aboutissant ainsi à une mort cellulaire apoptotique / The aim of this study was to investigate the anti-leukemic activity of UNBS1450, a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family, known to be a potent autophagy inducer in solid tumor cells. Interestingly, we hereby report for the first time that at low nanomolar concentrations, UNBS1450 induces apoptotic cell death in human leukemia cell lines. Subsequently, we have investigated the molecular mechanisms induced : Our results show that UNBS1450 inhibits NF-?B transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9 and 3/7, by decreasing the expression of anti-apoptotic Mcl-1 and by recruiting pro-apoptotic Bak and Bax eventually resulting in cell death

p53 is a critical tumour suppressor which acts to repair or remove abnormal cells and thus prevent cancer. Aberrant function of p53 is therefore a critical step in tumourigenesis and p53 is mutated in half of all cancers. Mutation of p53 leads to both a loss of normal wildtype function as well as the gain of oncogenic function. p53 is considered to be a promising therapeutic target and therapeutic strategies for targeting of the p53 pathway include: 1. Activation of wild-type p53 (wtp53) protein function, 2. Refolding of mutant p53 (mtp53) into the wtp53 conformation, 3. Reduction of mtp53 protein levels. In this work a number of small molecule screening assays were used to identify potentially novel regulators of both wtp53 and mtp53. Screening of a protein kinase inhibitor library for small molecules which can stimulate wtp53 activity identified the GSK3 pathway and a CDK pathway as dominant suppressors of wtp53 function. Screening of the library for inhibitors which reduce mtp53 protein levels led to the identification of two IKKβ inhibitors. The work then focused on investigating the effects of one of these compounds, IMD0354, on the mutant p53 pathway; with a specific focus on MDM2 as the most rapidly responding biomarker. IMD0354 is a well characterised inhibitor which has been shown to specifically inhibit IKKβ leading to the repression of the Nf-κB pathway. This study shows that IKKβ inhibition leads to the loss of a number of oncogenic proteins including mtp53, MDM2 and cyclin D. Mass-spectrometry based (ITRAQ) proteomic analysis was then employed to identify potential mediators and/or co-regulated factors in response to IKKβ-inhibition via IMD0354 treatment. This led to the identification of RPS3 as a potential negative regulator of MDM2 protein expression; the reduction in MDM2 protein in response to IMD0354 treatment is shown to be partially dependent on RPS3. Together this data has identified, using small molecule kinase inhibitor libraries: (i) dominant kinase signalling pathways that suppress wt-p53 and (ii) a dominant kinase signalling pathway that sustains expression of mutant p53 and MDM2 in cancer cell lines. This latter data supports further investigation to aid understanding of how the IKK signalling pathway cross-talks to the p53-MDM2 axis.

L'ajout simultané d'inhibiteurs des histones déacétylases (HDAC) au TNFα prolonge l'induction du facteur de transcription NF-kB en stabilisant l'activation du complexe IKK. Au cours de ce travail, l'effet de la Trichostatin A (TSA), un inhibiteur de HDACs à large spectre daction, sur les différentes voies d'activation du NF-kB a été étudié. L'effet de laddition simultanée de la TSA aux inducteurs de la voie classique (TNFα, PMA), de la voie alternative (Lymphotoxine β) et des voies alternatives (H2O2 et Pervanadate de Sodium) a été analysé. Le pervanadate de sodium (PV) est agent insulino-mimétique qui inhibe les tyrosines phosphatases et mime un stress oxydant. Ces études comparatives ont montré un prolongement de l'activité du NF-kB si la TSA est ajoutée simultanément au TNFα, PMA et au PV ; pas si elle est ajoutée à l'H2O2 ou à des agents induisant la voie alternative. Les mécanismes moléculaires sous-jacents aux prolongements de l'activation élicitée par le TNFα et le PV ont été étudiés en détails. Deux processus différents sont impliqués. La TSA ajoutée aux inducteurs de la voie classique prolonge l'activité du complexe IKK alors qu'ajoutée au PV elle induit un retard de synthèse important du mRNA de l'inhibiteur IkBα. De nombreuses expériences d'immuno-précipitation de la chromatine ont montré que l'ajout de la TSA au PV diminue (i) le recrutement de l'ARN polymérase II, (ii) l'acétylation et la phosphorylation de l'histone H3 sur la Lys14 et la Ser 10 respectivement, (iii) la phosphorylation sur la Ser 536 de p65 et (iv) le recrutement d'IKKα. Il est important de savoir qu'aucune de ces différences ne s'observent au niveau du promoteur d'Icam-1, un autre gène dépendant du NF-kB. Ces observations ont donc montré que l'effet de la TSA sur l'activation du NF-kB dépend non seulement du type de stimuli mais aussi du promoteur. Ce travail met en évidence un rôle inhibiteur des HDAC dans l'activation du NF-kB qui varie en fonction des stimuli.