Orientadores: Jacqueline Mendonça Lopes de Faria, José Butori Lopes de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T10:54:36Z (GMT). No. of bitstreams: 1 Duarte_DiegoAndreazzi_D.pdf: 28811769 bytes, checksum: 726e2f631efd3a14fedaf1f8148784aa (MD5) Previous issue date: 2014 / Resumo: A retinopatia diabética (RD) é uma doença devastadora que está entre as maiores causas de cegueira entre pessoas na idade adulta em todo o mundo. Considerada multifatorial e progressiva, a RD afeta células neurais e gliais, e também elementos vasculares da retina. Sabe-se que diversas vias estão envolvidas na patogênese da RD, no entanto, os mecanismos que levam a exacerbação da inflamação e morte de células gliais/neurais o que caracteriza a neurodegeneração da retina, ainda permanecem desconhecidos. Diante disso, a redução desses fatores tem sido extensivamente estudada como alvo no combate a RD. As Sirtuínas, histonas desacetilases dependentes de nicotinamida adenina dinucleotídeo (NAD+), atuam em resposta a vários estresses e atualmente tem sido relacionadas às importantes funções moleculares na regulação de várias doenças. Considerado um redox sensível, a SIRT1 pode estar reduzida em condição de doença, o que agravar ainda mais a situação patológica. No entanto, não se sabe ao certo o mecanismo de modulação e/ou atuação da SIRT1 frete às doenças neurodegenerativas, tais como a RD. No Artigo I, foram avaliados os possíveis efeitos protetores do cacau rico em polifenóis na retina diabética. As células Müller da retina (rMC-1) foram expostos por 24h à glicose normal (NG), alta glicose (HG) ou peróxido de hidrogênio (H2O2) e submetidas ao tratamento com cacau na presença ou não de um inibidor da SIRT1 ou siRNA. O estudo animal foi desenvolvido em ratos experimentalmente diabéticos induzidos por estreptozotocina e randomizado para receber tratamentos com cacau com baixa, intermediária, ou elevada dose de polifenol (0,12 mg; 2,9 mg; 22,9 mg/kg/dia) por gavagem durante 16 semanas. As células expostas a H2O2 ou HG apresentaram aumento de proteína acídica fibrilar glial (GFAP) e acetil-RelA/p65 e diminuição da atividade/expressão da SIRT1. Estes efeitos foram anulados pelo cacau, que diminuiu a produção de espécies reativas de oxigênio e reduziu a ativação da poli(ADP-ribose) polimerase-1 (PARP-1); melhorou os níveis intracelulares de NAD+ e consequentemente aumentou da atividade da SIRT1. As retinas dos ratos diabéticos exibiram os primeiros marcadores de retinopatia acompanhada pela eletrorretinografia prejudicada. A presença de diabetes levou a ativação da PARP-1 e diminuição dos níveis de NAD+, resultando em comprometimento da SIRT1. O aumento na acetilação do RelA/p65 levou na hiperexpressão do GFAP. A administração oral de cacau polifenol restaurou as alterações acima referidas. Este estudo revelou que o cacau enriquecido com polifenóis teve efeito protetor da retina diabética restabelecendo a via da SIRT-1. No Artigo II, foi investigado o possível efeito terapêutico de células derivadas de animais saudáveis (Dock7 m +/+ Leprdb db/m) e diabéticos (BKS.Cg-Dock7 m +/+ Leprdb/J, db/db) na retinopatia diabética (RD). Os camundongos db/db (espontaneamente diabéticos) com 8 semanas de idade foram randomizados para receber uma única injeção intravenosa de PBS ou células early outgrowth (EOCs) de doadores db/m ou db/db. Quatro semanas mais tarde, os animais foram sacrificados e os olhos enucleados. Para estudo in vitro, o meio condicionado das EOCs (EOC-CM) foi gerado a partir do cultivo de EOCs de animais db/m e db/db. As células rMC-1 foram expostas por 24h a NG ou HG e submetidas ao tratamento com db/m ou db/db EOC-CM, em presença ou não de um inibidor farmacológico (EX527) ou gênico (siRNA) da SIRT1. Nos ratos diabéticos, houve um aumento de marcadores de RD e do dano oxidativo, acompanhado por uma diminuição da proteína SIRT1 e seguido pelo aumento da acetilação da lisina-310 do complexo p65-NFkB. A terapia celular com EOCs reduziu significativamente todas as alterações mencionadas acima. As rMC-1 expostas a HG apresentaram aumento da expressão de GFAP, fator de crescimento do endotélio vascular e Nox4, acompanhado pelo aumento dos níveis de espécies reativas de oxigênio e acetil-lisina-310-p65-NFkB. Além disso, a expressão/atividade da SIRT1 foram reduzidas em ambiente diabético. O tratamento com EOC-CM impediu todas estas alterações. Este estudo demonstra que a capacidade parácrina das EOCs, na secreção de fatores, é eficaz no restabelecimento da via de SIRT1 retina, e assim, proteger a retina dos insultos diabéticos. Em resumo, a presente tese fornece evidências que tanto a administração oral do cacau enriquecido com polifenóis quanto à terapia celular com EOCs, conferem neuroproteção da retina aos insultos do diabetes. Portanto, intervenções que modulem a atividade das sirtuínas são promissoras no tratamento farmacológico da retinopatia diabética / Abstract: The diabetic retinopathy (RD) is a devastating disease and the principal cause of blindness among people in adulthood worldwide. The RD is considered a multifactorial and progressive disease, affecting neuronal and glial cells, and also vascular elements of the retina. It is known that several pathways are involved in the pathogenesis of RD, however, the mechanisms that lead to exacerbation of inflammation and death of glial/neuronal cell, characterizing retinal neurodegeneration, remain unknown. Therefore, the reduction of these factors have been extensively studied as a therapeutic target against RD. Sirtuin 1 (SIRT1), a family of histone deacetylase enzyme, acts in response to various stresses and, currently, has been related to important molecular functions in the regulation of various diseases. Considered a redox-sensitive, SIRT1 may be reduced under disease condition, whereby aggravate the pathological situation. However, is not known the mechanism of modulation/activity of SIRT1 in neurodegenerative diseases, such as RD. In the article I, were studies the possible protective effects of cocoa in the diabetic retina were assessed. rMCs exposed to NG, HG or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and siRNA. The experimental animal study was conducted in streptozotocin-induced diabetic rats and randomized to receive low, intermediate, or high polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12 mg/kg/day, 2.9 mg/kg/day, or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased GFAP and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased ROS production and PARP-1 activity, augmented the intracellular pool of NAD+, and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD+ levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of upregulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult. In the article II, were investigated the possible therapeutic effect of cells derived from control (db/m) and spontaneously diabetic (db/db) mice on diabetic retinopathy. The db/db mice with 8 weeks of age were randomized to receive a unique intravenous injection of PBS or 0,5x105 db/m EOCs or 0,5x105 db/db EOCs. Four weeks later, the animals were euthanized and the eyes enucleated. For in vitro study, EOC-CM was generated from db/m and db/db EOCs cultures. rMCs were exposed for 24h to NG or HG combined or not with db/m or db/db EOC-CMs. In diabetic rats, there was an increase of DR and oxidative damage markers, accompanied by decrease in SIRT1 protein followed by lysine-310-p65-NF?B acetylation. The treatment with cells from db/m significantly reduced all the above-mentioned, but interestingly the treatment with cells from db/db mice fully restored the above alterations to normal levels. rMCs exposed to HG displayed GFAP and VEGF expression up regulated, accompanied by increase in Nox4 expression and ROS levels, and acetyl-lysine-310-p65-NF?B. SIRT1 protein expression and activity were markedly reduced in diabetic milieu conditions. The treatment with both EOC-CMs prevented all these abnormalities, but db/db EOC-CM fully restored to NG conditions. This study demonstrates that endocrine capacity of EOCs is effective in improving retinal SIRT1 pathway thus protecting the retina from diabetic milieu insult. In summary, compelling novel evidence is provided herein that either through oral administration of polyphenol enriched cocoa or cell therapy with EOCs, conferred retinal neuroprotection against diabetic insults in animal models. The identification of SIRT-1 as a potential therapeutic target in the treatment of diabetic retinopathy may provide new perspective in the pharmacological treatment of this diabetic complication / Doutorado / Clinica Medica / Doutor em Clínica Médica

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

L'infection au virus de l'hépatite B (VHB) est étroitement liée au métabolisme énergétique hépatique. La réplication du virus est contrôlée en principal par des facteurs de transcription et récepteurs nucléaires tels que PPARa, HNF4a et Foxül, impliqués dans ce métabolisme. Ainsi, la réplication du virus est augmentée par la privation de nutriments et le stress énergétique en modèles cellulaires, et par le jeûne, en modèles murins. PGC-la, un régulateur majeur de la réponse métabolique adaptative au jeûne, est impliqué dans l'augmentation de la transcription du VHB par son interaction avec plusieurs facteurs de transcription. Il est connu que le récepteur des acides biliaires, FXRa, qui est capable d'activer le promoteur de Core du VHB, est co-activaté par PGC-la. Un autre acteur important dans l'adaptation métabolique à la privation d'énergie est la protéine déacétylase SIRTl. Lorsqu'il est activé, SIRTl hépatique est capable de désacétyler et activer autant PGC-la que FXRa. Ces données nous ont amenés à émettre l'hypothèse que SIRTl pourrait coopérer avec FXRa et PGC-la pour augmenter la transcription du VHB. Dans un premier travail, nous avons donc étudié le rôle de la coopération de ces trois facteurs métaboliques dans la réplication du virus. Ça nous a permis de décrire un nouveau réseau métabolique, composé de FXRa, PGC-la et SIRTl, qui régule l'activité transcriptionnelle du VHB. Nous avons montré que SIRTl augmente l'activité du promoteur de Core par l'intermède d'autre facteurs, parmi lesquels, FXRa. Nous avons en outre observé que la fonction de déacétylase de SIRTl était nécessaire pour l'amplification de l'effet de FXRa sur VHB promoteur de Core. Une autre cible de SIRTl, connue pour son activité co-activatrice sur FXRa, est PGC-la. Grâce à une série d'expériences de surexpression et suppression, nous avons montré que non seulement la co-activation de FXRa par PGC-la est potentialisée par SIRTl, mais la présence de PGC-la est nécessaire pour l'effet de SIRTl sur l'activation du promoteur de Core VHB induite par FXRa. Ces données suggèrent que FXRa, PGC-la et SIRTl coopèrent dans la modulation de l'activité transcriptionnelle du promoteur de Core. Nous avons ensuite confirmé nos observations initiales et avons montré que l'activation de l'axe SIRTl/PGC-la/FXRa induit la transcription de l'ARN de VHB dans des lignées cellulaires d'origine hépatique et non-hépatique. Ces résultats renforcent l'idée que la réplication du VHB peut être modulée en fonction de l'état nutritionnel. Les rapports des études précédentes menées in vitro et sur des modèles animaux suggèrent que la transcription du VHB est contrôlée de la même manière que les gènes de la néoglucogenèse. Notre hypothèse a été que chez l'homme, la réplication du VHB montrerait des fluctuations diurnes, selon les périodes de la journée de jeûne et de réalimentation. Le but de la deuxième étude a été donc de déterminer si la charge viral du VHB plasmatique montre des variations importantes tout au long du nichthemeron chez les patients chroniquement infectés par VHB, avec une réplication virale active [etc...] / Hepatitis B virus (HBV) infection is tightly linked with hepatic fuel metabolism. HBV replication depends on the activity of several liver-enriched nuclear receptors and transcription factors, such as PPARa, HNF4a, and Fox01, involved in the metabolic adaptive response to fasting. In the first part of our work, we identified a metabolic subnetwork that enhances the activity of HBV core promoter. FXRa (NR1H4), PPAR gamma coactivator 1a and SIRT1, the members of this regulatory axis, cooperate to increase HBV transcription. The three molecules are themselves key factors of liver metabolism, linking HBV replication to complex metabolic cues, such as energy status and nutrient availability during the fasting-refeeding cycles. We then observed the existence of a circadian cycle of HBV replication in humans, underlining the role of nutrient availability in the modulation of HBV replication, previously predicted by experimental models. The second part of the work focused on the plasma cell-free nucleic acids as potential biomarkers in chronic viral hepatitis. Due to the multiple links between HBV replication and cellular factors involved in fuel metabolism, we hypothesized that plasma mRNAs corresponding to these factors may constitute potential biomarkers for chronic hepatitis B. We successfully detected more than 30 plasma mRNA sequences corresponding to enzymes, transporters, nuclear receptors and transcription factors involved in fatty acids synthesis and oxidation, cholesterol synthesis, transport and excretion, and energy sensing and expenditure. The circadian variation and the multiple correlations in the expression patterns of these plasma transcripts are similar to those previously described in cells both in vitro and in vivo. This suggests that cell- free mRNAs may provide a "virtual biopsy" of the transcriptional status of the organism. Moreover, we found significant differences in the plasma mRNA profiles of HBV carriers compared with healthy controls, similar to those found in experimental models of infection, suggesting that these transcripts may also serve as biomarkers of liver disease. Further research is warranted to shed new light on the complex relationship between HBV life cycle and host lipid-carbohydrate-fuel metabolism and may lead to the identification of both actionable targets in antiviral therapy, and putative biomarkers in chronic hepatitis B

Le stress oxydant (SO) est un mécanisme commun à l’ischémie cérébrale et au traumatisme crânien qui entraîne notamment l’hyperactivation délétère de la poly(ADP-ribose)polymérase (PARP), une enzyme NAD+-dépendante. Cette dernière est impliquée dans le déficit neurologique et la lésion cérébrale consécutifs à ces pathologies. In vitro, l’hyperactivation de la PARP diminue le taux cérébral de NAD+, son substrat, et l’activité de la sirtuin 1 (SIRT1), une enzyme également NAD+-dépendante. L’activation de la SIRT1 est bénéfique au cours d’un SO in vitro. Si les effets bénéfiques de l’inhibition de la PARP ont été démontrés in vivo au cours d’un SO cérébral, l’implication de la SIRT1 ainsi que son rôle dans les effets de l’inhibition de la PARP n’ont pas été explorés. Dans la première partie de ce travail, nous avons mis en évidence qu’un modèle de SO cérébral induit in vivo chez le rat par une injection intrastriatale de malonate entraîne un SO prolongé, un déficit neurologique et une activation de la PARP associée à une diminution du NAD+. Dans la deuxième partie de ce travail, nous avons montré que le 3-aminobenzamide (3AB), un inhibiteur de la PARP, ne permet pas de s’opposer à la chute du NAD+ dans ce modèle, ce qui suggère que le NAD+ pourrait être consommé par d’autres enzymes NAD+-dépendantes, dont la SIRT1. L’inhibition de la PARP par le 3AB a permis d’augmenter le rapport activité/expression nucléaire de la SIRT1 et a entraîné sa translocation cytoplasmique au cours du SO. Un prétraitement par le SRT1720, un activateur spécifique de la SIRT1, diminue le déficit neurologique et la lésion striatale 6 heures après le SO cérébral, ce qui suggère que l’activation de la SIRT1 est bénéfique dans les conséquences d’un SO cérébral in vivo. L’association de l’inhibiteur de la PARP avec l’activateur de la SIRT1 (3AB+SRT1720) n’a pas potentialisé les effets protecteurs de chaque monothérapie. L’EX527, un inhibiteur de la SIRT1, ne modifie pas le déficit et la lésion. En revanche, l’association de l’inhibiteur de la PARP avec l’inhibiteur de la SIRT1 (3AB+EX527) supprime la récupération neurologique ainsi que la réduction de la lésion, induites par l’inhibition de la PARP seule (3AB). Ces données suggèrent que l’activation de la SIRT1 est impliquée dans les effets bénéfiques de l’inhibition de la PARP in vivo au cours d’un SO cérébral. En conclusion, l’ensemble de ce travail a permis une meilleure caractérisation de la PARP et de la SIRT1 au cours d’un SO cérébral in vivo. La SIRT1 pourrait constituer une cible pharmacologique pour le traitement des pathologies cérébrales au cours desquelles un SO est présent. De plus, nous avons montré que les effets bénéfiques de l’inhibition de la PARP sur les conséquences fonctionnelles et histologiques induites par le SO cérébral sont liés à l’activation de la SIRT1. / Oxidative stress (OS) is involved in cerebral ischemia and traumatic brain injury and results in deleterious activation of poly(ADP-ribose)polymerase (PARP), an NAD+-dependant enzyme. PARP is implicated in neurological deficit and brain injury post-ischemia and post-trauma. In vitro, PARP overactivation reduced both brain NAD+ levels, its substrate, and activity of sirtuin 1 (SIRT1), an other NAD+-dependant enzyme. SIRT1 activation is beneficial during in vitro OS. Even if the beneficial effects of PARP inhibition have been demonstrated, SIRT1 involvement during in vivo cerebral OS and its role in the beneficial effects of PARP inhibition have not been studied.In the first part, we demonstrated that in vivo cerebral OS induced by intrastriatal injection of malonate in rat promoted prolonged OS, neurological deficit, PARP activation and NAD+ decrease. In the second part, we showed that 3-aminobenzamide (3AB), a PARP inhibitor, did not reduce NAD+ loss, suggesting that NAD+ could be consumed by other NAD+-dependant enzymes, including SIRT1. The PARP inhibitor increased the nuclear SIRT1 activity/expression ratio and induced its cytoplasmic translocation during OS. SRT1720, a specific SIRT1 activator, reduced both neurological deficit and striatal lesion 6 hours after cerebral OS, suggesting that SIRT1 activation is beneficial on in vivo OS consequences. The combination of the PARP inhibitor with the SIRT1 activator (3AB + SRT1720) did not potentiate the neuroprotective effects of each strategy. EX527, a SIRT1 inhibitor, did not affect OS-induced deficit and lesion. However, association of the PARP inhibitor with the SIRT1 inhibitor (3AB + EX527) suppressed the neurological recovery and the reduction of lesion induced by 3AB alone. Our data suggested that SIRT1 activation is involved in the neuroprotective effects of PARP inhibition during in vivo cerebral OS. In conclusion, our work led to a better characterization of PARP and SIRT1 during in vivo cerebral OS. SIRT1 is a potential pharmacological target for the treatment of brain pathologies in which OS is present. In addition, SIRT1 activation is involved in the beneficial effects of PARP inhibition on functional and histological cerebral OS consequences

Au cours des maladies cardiovasculaires (MCV), il peut se produire divers problèmes de santé, telle que l’insuffisance cardiaque ou encore l’HTA. Ces phénomènes se caractérisent, entre autres, par une augmentation de synthèse d’endotheline-1 (ET-1), un neuropeptide synthétisé par les cellules endothéliales ayant un effet vasoconstricteur sur les cellules musculaires lisses vasculaires (CMLV). Ainsi, la surexpression de ce vasopeptide, mène à terme, au maintien de l’HTA aggravée des sujets, précédée ou concomitante à l’athérosclérose ou à la resténose, cliniquement illustrées par une prolifération et une migration anormale des CMLV de la media vers l’intima des vaisseaux sanguins. Parallèlement, il a été observé que la protéine sirtuine-1 (Sirt-1), membre de la famille des protéines histones déacétylases (HDAC), présente des propriétés anti-athérosclérotiques par sa capacité d’atténuer la prolifération et la migration des CMLV. Des travaux récents ont aussi montré qu’au cours de l’HTA la protéine Sirt-1 est faiblement exprimée dans les CMLV. Son implication dans le développement des pathologies vasculaires semble apparente, mais des études demeurent nécessaires pour décrire son rôle exact dans la pathogenèse des MCV. Dans cette optique, l’objectif de cette étude a été d’observer la variation d’expression de Sirt-1 dans les CMLV, isolées de l’aorte ascendante de rat, en réponse à l’ET-1. On a remarqué qu’une heure de stimulation des CMLV avec l’ET-1 induit une diminution de l’expression de Sirt-1 via l’activation des récepteurs ETA. Ces résultats suggèrent que la capacité d’ET-1 à atténuer l’expression de Sirt-1 serait un éventuel mécanisme d’action avec des effets favorisant les MCV. / Cardiovascular diseases (CVD) are associated with several vascular dysfunctions such as heart failure and hypertension. These phenomena cause increased synthesis of endothelin-1 (ET-1), a neuropeptide, synthesized by endothelial cells which has vasoconstrictor action on vascular smooth muscle cells (VSMC). Overexpression of this vasopeptide leads eventually to hypertension (HTA). This usually happen after atherosclerosis or restenosis, leading to proliferation and migration of VSMC from media to intima. It was shown that during atherosclerosis, the protein sirtuin-1 (Sirt-1), a member of protein histone deacetylases (HDAC), has an anti-atherosclerotic effect due to its ability to diminish proliferation and migration of VSMC. It has also been observed that during hypertension, Sirt-1 was poorly expressed in VSMC. Its role in vascular pathophysiology remains sparsely studied, therefore it’s essential to explore it. In the present study we investigated the expression of Sirt-1 in VSMC isolated from the ascending aorta of rats, in response to ET-1 stimulation. We observed that Sirt-1 expression decreases after 1 hour of stimulation by ET-1 via ETA receptors. In summary, these results suggest that the ability of ET-1 to attenuate Sirt-1 expression in VSMC, may be a potential mechanism for promoting CVD.

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

L'acétylation des protéines constitue l’une des plus importantes modifications post-traductionnelles (PTMs). Elle intervient dans de multiples processus bologiques et physiopathologiques tels que, l’activité transcriptionnelle, l'apoptose, la régulation des voies métaboliques, les cancers, les maladies inflammatoires et cardiovasculaires. Face à l’importance de l’acétylation des protéines, il apparaît donc indispensable de bien comprendre les mécanismes qui y sont associés, et donc, de pouvoir identifier et quantifier les protéines acétylées à partir du protéome complet d’échantillons complexes tels que des extraits cellulaires ou tissulaires. La spectrométrie de masse est une technique de choix pour de telles études, car elle permet d’identifier les protéines et les sites d’acétylation, mais aussi de les quantifier en l’associant à des techniques de quantification (label free, SILAC, iTRAQ/TMT, AQUA). Malheureusement, ces méthodes ne ciblent pas particulièrement les acétylations et requièrent l’utilisation de techniques d’enrichissement ou de fractionnement qui ne sont dédiées qu’à certains types d’acetylation : les N-ter et K-acetylation. Aucun enrichissement n’est disponible pour les O- acétylations et ces méthodes d’enrichissement ne sont pas toujours compatibles avec les techniques de quantification citées ci-dessus. Pour améliorer la détection et la quantification des acétylations, nous proposons la méthode RAQIAT (Relatif Absolute Quantification Isobaric Affinity Tag) qui se résume en trois grandes étapes: i) Le blocage des fonctions amines libres à l'aide de la di-méthylation réductrice, ceci empêchera ces dernières de réagir avec le réactif RAQIAT, ii) La désacétylation des lysines acétylées pour permettre une quantification sélective des acétylations, iii) Le marquage des amines primaires précédemment désacétylées dans l’étape 2 par le réactif RAQIAT pour permettre leurs identifications et quantifications. Ce manuscrit a porté en partie sur les deux premières étapes de la méthode RAQIAT.Dans la première étape, les échantillons de protéines de levure ont été digérés puis di-méthylés et fractionnés par OFFGEL en 24 fractions. Ensuite, chacune de ces 24 fractions OFFGEL a été soumise à un fractionnement nano-RPLC et analysée par MALDI TOF/TOF (4800 MALDI-TOF/TOF, Sciex). En parallèle, la même expérience a été réalisée, cette fois-ci sans di-méthylation. L'analyse des données a été réalisée en utilisant le logiciel Mascot comme moteur de recherche.L’efficacité de la réaction de di-méthylation démontrée, nous avons montré que sans réaliser la di-méthylation réductrice 164 sites acétylés ont pu été identifiés alors que 385 sites acétylés distincts ont été identifiés avec la di-méthylation réductrice. De plus, l'amélioration de la détection de l'acétylation en utilisant la méthode de di-méthylation a été observée pour chacune des différents types acétylations: N-ter, K- et O-acétylation.Dans la deuxième étape, nous avons présenté des résultats préliminaires de déacétylation par la sirtuine 1 en présence du peptide de la p53 (Ac-Arg-His-Lys-Lys-(Ac)-AMC) connu comme étant un substrat de cette enzyme. Nous avons observé la formation d’un peptide non acétylé, suggérant une déacétylation de ce peptide acétylé de p53. Cependant, la formation de cet ion étant très faible et l’ion acétylé étant fortement préservé, nous en avons conclu que l’efficacité de la déacétylation du peptide de p53 n’était pas suffisante pour l’intégrer à la méthode RAQIAT. / Protein acetylation is one of the most widespread post-translational modifications which is involved in many cellular physiologies and pathologies such as cancers. Regarding the important biological effect of protein acetylation and a non-negligible number of proteins bearing this PTM, several methods emerged last decade to investigate such PTM. But the detection of acetylations and their quantification are still limited and enrichment method allowing a better detection of acetylation target mostly one kind of acetylation (K-acetylation). To improve the detection of the three kind of acetylation (N-ter, K, and O-) and their quantification, we propose the RAQIAT method (Relative Absolute Quantification Isobaric Affinity Tag), based on protein digestion followed by 3 steps : i) a protection of free primary amines at N-ter, lysine (i.e. primary amine not bearing PTM) based on a reductive di-methylation strategy ii) a deacetylation of acetylated residues to obtain free primary amine corresponding to peptides previously acetylated iii) a RAQIAT labeling on the free primary amine obtained in the previous step to allow the enrichment of peptides previously acetylated and their quantifications. Herein, we present the investigation of the two first steps of RAQIAT method.In the first step, we evidenced that the reductive di-methylation strategy improved the detection of the three kind of acetylation: N-ter, K- and O- acetylations. Yeast protein samples were digested with trypsin prior di-methylation of resulting peptide mixture. Then, di-methylated peptide mixtures were fractionated by OFFGEL and reverse phase liquid chromatography followed by MALDI-TOF/TOF mass spectrometry analysis. Data analysis was performed by using Mascot as search engines.Our results showed that OFFGEL fractionation is a useful step to increase detection of acetylations. Moreover, we showed that our di-methylation treatment improved significantly detection of acetylation. Indeed, after di-methylation treatment, 385 unique acetylated sites were identified while 164 unique acetylated peptides were detected without di-methylation treatment. The improvement of acetylation detection using our di-methylation strategy is observed for each of acetylations: N-ter, K- and O-acetylations. Thus, this new proteomic method is promising to enhance N-ter, K- and O-acetylation detection.In the second step, we presented preliminary results of deacetylation by sirtuin 1 in the presence of p53 peptide (Ac-Arg-His-Lys-Lys- (Ac) –AMC. However, the low deacetylation efficiency of the p53 peptide observed, conclude that is not suitable to applicate into RAQIAT Method

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

Yes / The NAD(+)-dependent deacetylase SIRT1 is involved in diverse cellular processes, and has also been linked with multiple disease states. Among these, SIRT1 expression negatively correlates with cancer survival in both laboratory and clinical studies. Active regulator of SIRT1 (AROS) was the first reported post-transcriptional regulator of SIRT1 activity, enhancing SIRT1-mediated deacetylation and downregulation of the SIRT1 target p53. However, little is known regarding the role of AROS in regulation of SIRT1 during disease. Here, we report the cellular and molecular effects of RNAi-mediated AROS suppression, comparing this with the role of SIRT1 in a panel of human cell lines of both cancerous and non-cancerous origins. Unexpectedly, AROS is found to vary in its modulation of p53 acetylation according to cell context. AROS suppresses p53 acetylation only following the application of cell damaging stress, whereas SIRT1 suppresses p53 under all conditions analysed. This supplements the original characterization of AROS but indicates that SIRT1 activity can persist following suppression of AROS. We also demonstrate that knockdown of AROS induces apoptosis in three cancer cell lines, independent of p53 activation. Importantly, AROS is not required for the viability of three non-cancer cell lines indicating a putative role for AROS in specifically promoting cancer cell survival.

Dans les pays industrialisés, les rétinopathies ischémiques proliférantes telles que la rétinopathie diabétique et la rétinopathie du prématuré sont les principales causes de cécité chez les individus en âge de travailler et la population pédiatrique. Ces pathologies sont caractérisées par une dégénérescence microvasculaire initiale suivie d’une hyper-vascularisaton compensatoire disproportionnée et pathologique. Les sirtuines constituent une importante famille de protéines impliquées dans le métabolisme et la réponse au stress. Plus particulièrement, sirtuine 3 (SIRT3) est une déacétylase mitochondriale primordiale qui agit au cœur du métabolisme énergétique et de l’activation de nombreuses voies métaboliques oxydatives. Nos résultats démontrent pour la première fois qu’une déficience en SIRT3 diminue la sévérité des lésions vasculaires dans le modèle murin de rétinopathie induite par l’oxygène (OIR). En plus de stimuler l’angiogénèse, l’absence de SIRT3 est aussi associée à une augmentation de la glycolyse, possiblement en activant la famille de gènes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). Nous suggérons que le manque de SIRT3 est impliqué dans l’effet Warburg et procure ainsi un avantage prolifératif et protecteur dans l’OIR. La présente étude propose SIRT3 comme nouvelle cible thérapeutique potentielle dans la rétinopathie du prématuré, une maladie dont les complications désastreuses persistent tout au long de la vie. / Proliferative ischemic retinopathies such as proliferative diabetic retinopathy and retinopathy of prematurity (ROP) are the leading causes of blindness in working age and pediatric populations in industrialized countries. These pathologies are characterized by an initial microvascular degeneration followed by a disproportionate compensatory but pathological hyper-vascularization mounted by the hypoxic and energy deficient retina in an attempt to reinstate metabolic equilibrium. Sirtuins are an important family of protein involved in metabolism and stress response. Sirtuin 3 (SIRT3) in particular is a major mitochondrial deacetylase central to energy metabolism and the regulation of many oxidative pathways. For the first time, our results show that a lack of SIRT3 decreases the severity of vascular lesions in the oxygen-induced retinopathy (OIR) mouse model. Deficiency in SIRT3 not only stimulates angiogenesis, but also increases glycolysis, possibly through indirect activation of the gene family 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). We suggest that a lack of SIRT3 is involved in the Warburg effect and therefore confers a proliferative advantage that is protective in OIR. The present study puts forward SIRT3 as a new potential therapeutic target for ROP, a disease leading to life-long vision complications.