Oxidative stress is one of the major characteristics in Alzheimer’s disease, and converging evidence indicates that cysteine S-nitrosylation might be related in AD pathology. My results demonstrated exogenous S-nitrosoglutathione was able to S-nitrosylate vAChT, vMAT2, vGluT1 and vGluT2. S-nitrosylation of these vesicular transporters inhibited the uptake of [3H]acetylcholine, [3H]dopamine and [3H]glutamate respectively. APP/PS1 transgenic mice were used to investigate neurotransmission dysfunctions of Alzheimer’s disease. Global protein S-nitrosylation was increased in the 9 and 12 month APP/PS1 mice. Further investigation demonstrated an increase of vAChT and vGluT1 S-nitrosylation in frontal cortex of 6, 9 and 12 month APP/PS1 mice and an increased vAChT and vGluT1 S-nitrosylation was found in hippocampus of 3 month APP/PS1 mice. These findings together suggest that S-nitrosylation of vesicular transporters inhibits the uptake of neurotransmitters, and S-nitrosylation of vAChT and might be associated with the neurotransmission dysfunction of acetylcholine and glutamate in Alzheimer’s disease.

Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.

<p>S-nitrosylation, the prototypic mechanism of redox-based signal transduction, involves the covalent attachment of a nitrogen monoxide group to a Cys-thiol side chain. S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions and protein stability. The primary focus of my dissertation was to solve a problem of great importance in the field of S-nitrosylation, which is, to identify denitrosylase(s) i.e., enzymes that remove NO groups from S-nitrosothiols. Recent progress in elucidating the cellular regulation of S-nitrosylation has led to the identification of two physiologically relevant denitrosylating activities that remove the NO group from S-nitrosylated substrates. Thioredoxin/thioredoxin reductase (Trx system) functions as an NADPH-dependent denitrosylase across a broad range of S-nitrosylated proteins (SNO-proteins). S-nitroso-glutathione reductase (GSNOR), which is highly conserved across phylogeny, metabolizes GSNO utilizing NADH as a reducing coenzyme, thereby shifting equilibria between GSNO and SNO-proteins. This dissertation describes the discovery of two novel denitrosylases: one from yeast and the other from mammals. Using technique of column chromatography we have purified these novel denitrosylases to homogeneity and have demonstrated a principal contribution of these enzymes towards S-nitrosothiol metabolism.</p> / Dissertation

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Chez les plantes, l'oxyde nitrique (NO) est impliqué dans de nombreux processusbiologiques tels que la germination ou le développement racinaire et intervient dans les réponses àdivers stress biotiques ou abiotiques. Ainsi, en situation de stress en fer, la production de NOconstitue un événement précoce dans la voie de signalisation qui aboutit à l'induction desferritines. Les cibles du NO demeurent toutefois mal connues, mais il est établi qu'un effet majeurest la S-nitrosylation de cystéines dans les protéines. Dans ce travail, nous avons cherché àidentifier les protéines (et les cystéines) qui constituent les cibles moléculaires du NO dans deuxsituations de stress abiotique chez Arabidopsis thaliana. Dans ce but, une démarche deprotéomique post-traductionnelle dédiée, fondée sur la méthode classique dite du «Biotin-switch»(BS), a été privilégiée pour l'identification des protéines nitrosylées. Par ailleurs, afin de pouvoirévaluer les variations de nitrosylation et analyser des réponses physiologiques, nous avonsintroduit une dimension quantitative en combinant le BS à un marquage des thiols par des réactifsdifférant par la présence d'isotopes lourds (Isotope coded affinity tag, ICAT). La méthode ainsidéveloppée (BS-ICAT) a permis de caractériser des variations de nitrosylation de protéines lorsd'un stress ferrique et d'un stress salin. A côté de l'identification de cibles potentielles du NO, lesrésultats ont également attiré l'attention sur certaines limites du BS. Sur cette base, la méthodeBS-ICAT a été utilisée pour revisiter quantitativement le BS. Nous avons montré que le blocagedes thiols libres, étape initiale-clé fondant le BS, n'est que partiel et conduit à l'apparition de fauxpositifs. Simultanément, de nouveaux contrôles de spécificité ont été éprouvés. La combinaisonBS-ICAT constitue l'une des toutes premières tentatives pour la caractérisation quantitative àlarge échelle de réponses de nitrosylation. A côté d'un premier répertoire de sites de Snitrosylationchez les plantes et de l'identification de candidats potentiellement impliqués dans lesréponses aux stress en fer et en sel, l'analyse quantitative permet de proposer une utilisation du BSintégrant ses limites de façon plus contrôlée. Cette analyse souligne le besoin de méthodesalternatives où le marquage soit réalisé directement sur les nitrosothiols. / In plants, nitric oxide (NO) is involved in many biological processes such as germination or roots development and in responses to various biotic and abiotic stresses. Thereby, in iron stress situations, NO production is the earliest signaling pathway event leading to ferritins induction. NO targets remain largely unknown but it is now stated that cysteine S-nitrosylation in proteins is the main NO consequence. The present work aims at identifying NO target proteins in Arabidopsis thaliana in the context of two abiotic stresses. For this purpose, a posttranslational proteomics approach based on the classical “Biotin-Switch” method (BS) was favored to identify nitrosylated proteins. Moreover, in order to estimate changes in nitrosylation and analyze physiological responses, a quantitative dimension combining the BS and a differential isotope based labeling of thiol with the ICAT reagents (Isotope Coded Affinity Tag) was introduced. This method named BS-ICAT allowed us to characterize quantitative variations of S-nitrosylation during an iron and a salt stress. Beside the identification of potential NO targets, our results highlighted limitations of BS method through incomplete free thiol blockage, the key initial step in BS, leading to false positive identifications. Simultaneously, new control have been introduced to test the specificity of the labeling. The combination of BS and ICAT is one the first attempt to quantitatively characterize the NO response at a large scale. This quantitative analysis results in one of the first repertoire of S-nitrosylation sites in plant proteins under abiotic stress and highly suggests a careful use of BS under strict control conditions. Moreover this analysis re-enforces the emerging need for alternative methods where the labeling molecules react directly with the nitrosothiols.

La régulation de la photosynthèse est cruciale pour les organismes photoautotrophes et est habituellement opérée par la modulation de l'absorption de la lumière ou par la réorientation des électrons vers des puits alternatifs afin de redistribuer l'énergie entre plusieurs voies métaboliques. Parmi les différents mécanismes décrits, le remodelage de l'appareil photosynthétique est crucial dans des conditions de carences nutritives ou de fluctuations de la lumière. Il est bien connu que l'oxyde nitrique (NO) joue un rôle de signalisation dans de nombreuses réponses au stress abiotique, agissant comme second messager et / ou modifiant les protéines cibles par des modifications post-traductionnelles redox. Sa participation a été récemment décrite au cours de la carence en azote chez Chlamydomonas reinhardtii. Ce travail se concentre sur le remodelage de l'appareil photosynthétique lors de la carence en soufre et lors des fluctuations de lumineuses chez Chlamydomonas reinhardtii, avec un intérêt particulier pour la voie de signalisation impliquée dans ces réponses. Tout d'abord, nous avons caractérisé la carence en soufre en conditions d’hétérotrophie ou de photo-autotrophie. En faible lumière ou à l’obscurité, l'inactivation photosynthétique est obtenue grâce à la dégradation spécifique de la Rubisco et du cytochrome b6f et ne se produit qu'en présence de carbone réduit dans le milieu. Nous avons également montré une forte production de NO après le début de la carence, avec des sondes fluorescentes sensibles au NO visualisées par microscopie confocale. Nous fournissons des preuves pharmacologiques que la production de NO intracellulaire régit cette voie de dégradation. En outre, ici, nous fournissons des preuves claires de l’existence d’un circuit régulateur qui contrôle la traduction cytosolique du LHCII en réponse à des changements de quantité de lumière. Ce circuit nécessite la protéine de liaison à l'ARN cytosolique NAB1 pour réprimer la traduction de certains ARNm de LHCII. La nitrosylation spécifique de la Cys-226 diminue l'activité de NAB1 et a été démontrée in vitro et in vivo. La forme moins active et nitrosylée de NAB1 se trouve dans les cellules acclimatées à un apport de lumière limité, ce qui permet l'accumulation de protéines des antennes et la capture efficace de la lumière. En revanche, une intensité lumineuse plus élevée provoque la dénitrosylation de NAB1, activant ainsi la répression de la synthèse des protéines LHCII et diminuant ainsi la pression de la lumière au niveau du PSII. La dénitrosylation de NAB1 est efficacement réalisée par le système thiorédoxine cytosolique in vitro. À notre connaissance, NAB1 est le premier exemple de dénitrosylation induite par un stimulus dans le contexte de l'acclimatation photosynthétique. Dans l’ensemble, nos données suggèrent un rôle pivot pour la signalisation NO dans le contrôle des réponses au stress environnemental. / The regulation of photosynthesis is crucial for photoautotrophic organisms and is usually operated by the modulation of light absorption or by redirection of electrons towards alternative sinks, in order to redistribute energy among several metabolic pathways. Between different mechanisms described, the remodeling of the photosynthetic apparatus is crucial under conditions of nutrient starvation or light fluctuations. It is well known that nitric oxide (NO) plays a signaling role in many abiotic stress responses, acting as a second messenger and/or modifying target proteins through redox post translational modifications. Its involvement has been recently described during nitrogen starvation in Chlamydomonas reinhardtii. This work focuses on the remodeling of the photosynthetic apparatus upon sulfur starvation and light fluctuations in Chlamydomonas reinhardtii, with particular interest for the signaling pathway involved in the responses. First we characterized sulfur starvation under heterotrophy and photo-autotrophy. Photosynthetic inactivation under low light and darkness is achieved through specific degradation of Rubisco and cytochrome b₆f and occurs only in the presence of reduced carbon in the medium. We have also shown a strong NO production after the onset of starvation, with NO-sensitive fluorescence probes visualized by confocal microscopy. We provide pharmacological evidence that intracellular NO production governs this degradation pathway using NO scavengers, NO synthesis inhibitors and NO donors. Furthermore, here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. This circuit requires the cytosolic RNA-binding protein NAB1 to repress translation of certain LHCII mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light harvesting proteins and efficient light capture. In contrast, elevated light supply causes NAB1 denitrosylation, thereby activating the repression of light-harvesting protein synthesis and decreasing the light pressure at the level of PSII. Denitrosylation of NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. Taken together, our data suggest a pivotal role for NO-signaling in the control of environmental stress responses.

L’effet anti-tumoral d'un lipide A, l’OM-174 (partie lipidique des lipopolysaccharides) a été étudié dans un modèle de cancer mammaire chez la souris. In vivo, l’OM-174 augmente la survie de la souris alors qu’in vitro il n'est pas toxique pour les cellules cancéreuses. L’OM-174 se lie au récepteur TLR4 des cellules immunitaires induisant la production de cytokines comme l’IFNγ. In vitro, l’association de cette cytokine au lipide A est cytotoxique. L’objectif de cette thèse est d’en analyser les mécanismes moléculaires. Nous avons montré, aussi bien in vitro qu’in vivo, que la cytotoxicité du lipide A/IFNγ est dépendante du TLR4, du récepteur à l’IFNγ et de l’expression de la NOS II. Nous avons également montré que les espèces radicalaires, NO et anion superoxyde formant le peroxynitrite jouent un rôle crucial dans cette cytotoxicité. L’origine de ces espèces radicalaires se trouve être la NOS II selon un processus de découplage enzymatique. Nous avons également cherché d’autres mécanismes associés pouvant expliquer la cytotoxicité du lipide A/IFNγ. Nous avons ensuite montré que le NO est capable de réagir avec les résidus cystéine de certaines protéines, un processus appelé S-nitrosylation. Une analyse protéomique nous a permis d’identifier au moins une dizaine de protéines qui sont S-nitrosylées dans les cellules cancéreuses mammaires en réponse au lipide A/IFNγ. Nous avons étudié l’impact de cette modification sur la fonction d’une des ces protéines, l’enzyme de conjugaison E2 de l’ubiquitine Ubc13, une protéine impliquée dans la prolifération et la survie cellulaire. Nous avons confirmé la nitrosylation d’Ubc13 et identifié la cystéine 87 comme cible du NO. L’expression d’une forme mutée d’Ubc13 (remplacement de la cystéine 87 par une alanine) inhibe l’auto-ubiquitination de l’enzyme et sa capacité à ubiquitiner une de ses cibles IkBα. Nous avons montré que la S-nitrosylation d’Ubc13 induit sa migration vers le noyau et rend les cellules plus sensibles à l’effet cytotoxique du lipide A/IFNγ. En résumé, nos résultats révèlent un rôle majeur et insoupçonné de la NOS II et du NO dans l’effet antitumoral du lipide A OM-174 dans un modèle de cancer mammaire chez la souris ouvrant la voie pour la conception de nouveaux traitements anticancéreux. / The anti -tumor effect of a lipid A, OM -174 (lipid portion of LPS) was studied in a model of breast cancer in mice. In vivo, OM- 174 increases the survival of mice whereas in vitro it is not toxic to cancer cells. OM -174 binds to TLR4 immune cells inducing the production of cytokines such as IFNγ. In vitro, the combination of IFNγ to lipid A is cytotoxic. The objective of this thesis is to analyze those molecular mechanisms. We have shown both in vitro and in vivo that the cytotoxicity of the lipid A / IFNγ is dependent of TLR4 and of the receptor for IFNγ, and the NOS II expression. We also showed that the radical species, NO and superoxide anion forming peroxynitrite play a crucial role in this cytotoxicity. The origin of these radical species is being NOS II enzyme in a process of decoupling. We also looked for other associated mechanisms that may explain the cytotoxicity of lipid A / IFNγ. We then showed that NO is able to react with the cysteine residues of certain proteins, a process called S- nitrosylation. A proteomic analysis allowed us to identify at least a dozen proteins that are S- nitrosylated in breast cancer cells in response to lipid A / IFNγ. We studied the impact of this change on the basis of one of these proteins, the E2 conjugating enzyme UBC13 ubiquitin, a protein involved in cell proliferation and survival. We confirmed the UBC13 nitrosylation on cysteine 87 and identified as a target of NO. The expression of a mutant of UBC13 (replacement of cysteine 87 with alanine) forms inhibits the auto-ubiquitination of the enzyme and its ability to ubiquitinylated one of its targets IkBα. We have shown that S- nitrosylation of UBC13 induced its translocation to the nucleus and greater sensitivity to the cytotoxic effect of lipid A / IFNγ in cells. In summary, our results reveal an important and unexpected role of NOS II and NO in the antitumor effect of lipid A OM- 174 in a model of breast cancer in mice opening the way for the development of new cancer treatments.

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS’s cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2-and peroxynitrite (ONOO-). Previous work in our and other’s labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein we show that recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We also see increased tumor cell death measured by in vitro clonogenic assay, as well as decreased metabolic uptake in Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) induced colorectal cancer in vivo measured by [18F]-fluorodeoxyglucose ([18F]-FDG) positron emitted topography (PET) imaging. We believe by recoupling NOS both in vivo and in vitro we are modulating Wnt signaling via Akt and GSK-3β. Lastly, we conducted studies to determine a mechanistic explanation of how tumor cells maintain a decreased BH4:BH2 ratio.

Ao longo dos últimos anos, o radical livre gasoso óxido nítrico (NO) vem ganhando destaque como uma importante molécula sinalizadora em respostas fotomorfogênicas em plantas. Sua produção e dagradação parecem incluir uma diversificada gama de rotas bioquímicas, entretanto, a importância relativa de cada um dos sistemas capazes de regular sua disponibilidade e toxidade nos tecidos vegetais ainda permanece pouco compreendida. Dentre as possíveis rotas de conjugação e degradação do NO em tecidos vegetais, postula-se que a glutationa (GSH) desempenhe um papel de destaque no armazenamento desse radical livre por meio da formação reversível da S-nitrosoglutationa (GSNO), sendo possível sua subsequente degradação através da ação da enzima S-nitrosoglutationa redutase (GSNOR). No presente trabalho investigamos a influência da luz sobre o metabolismo de NO em duas etapas de desenvolvimento vegetal caracterizados pela ocorrência de eventos de diferenciação plastidial: (I) o desestiolamento de plântulas e (II) o amadurecimento de frutos carnosos de tomateiro (Solanum lycopersicum). Além do genótipo selvagem Micro-Tom (MT), também foram utilizados os mutantes fotomorfogênicos aurea (au) e high pigment 1 e 2 (hp1 e hp2). Durante o desestiolamento das plântulas de tomateiro constatou-se um incremento progressivo tanto nos teores endógenos quando nas taxas de degradação de NO, bem como na atividade da enzima GSNOR. Sob condições luminosas similares, mutantes com respostas exageradas à luz apresentaram incrementos ainda mais evidentes nesses parâmetros do que aqueles observados no genótipo selvagem. A aplicação de inibidores de S-nitrosilação de proteínas, bem como a avaliação do conteúdo de espécies reativas de oxigênio (ROS) indicaram que tanto a formação de S-nitrosotiois quanto a interação do NO com ROS contribuíram para a determinação da capacidade de remoção de NO nos tecidos fotossinteticamente ativos de tomateiro. Em frutos, observou-se uma correlação positiva entre a atividade da enzima nitrato redutase (NR) e o padrão temporal de produção de NO, uma vez que ambos os parâmetros apresentaram maiores níveis em frutos imaturos. O amadurecimento desses frutos foi acompanhado por uma diminuição transitória dos conteúdos de NO ao passo que as taxas de degradação de NO mantiveram-se bastante reduzidas durante todo o processo de amadurecimento, sugerindo a existência de um estoque de NO na forma de GSNO ou algum outro S-nitrosotiol. A sinalização luminosa influenciou positivamente tanto a produção quanto a degradação de NO em frutos imaturos de tomateiro. Em conjunto, os resultados obtidos permitem concluir que o metabolismo do NO em tomateiro é fortemente controlado pela luz, a qual é capaz de modular conjuntamente as taxas de produção e degradação desse importante composto sinalizador. / In recent years, the gaseous free radical nitric oxide (NO) has emerged as an important signaling molecule in plant photomorphogenic response. NO production and degradation seems to include a wide range of biochemical routes; however, the relative importance of which one of the systems capable of regulating NO availability and toxicity in plant tissues remains elusive. Among all potential NO degradation and conjugation routes in plant tissues, it has been suggested that gluthathione (GSH) plays a key role in NO storage due to the formation of S-nitrosogluthathione (GSNO), being possible its subsequent degradation by the action of enzyme S-nitrosoglutathione reductase (GSNOR). In this work, we have investigated the light influence on NO metabolism during two plant developmental events characterized by the occurrence of plastidial differentiation: (I) seedling de-etiolation and (II) fruit ripening of tomato (Solanum lycopersicum). Besides the wild-type Micro-Tom (MT) genotype, the tomato photomorphogenic mutants aurea (au) and high pigment 1 and 2 (hp1 and hp2) were also employed in this study. During the de-etiolation of tomato seedlings, a progressive increment was observed in the NO endogenous levels and degradation rates as well as in the GSNOR activity. Under similar light conditions, light hypersensitive mutants exhibited more conspicuous increases in these parameters than those detected in the wild-type genotype. Feeding protein S-nitrosylation inhibitors and measurements of reactive oxygen species (ROS) production indicated that both S-nitrosothiols formation and NO interaction with ROS may to contribute for determining the NO removal capacity in photosynthetically active tissues of tomato. In fruits, a positive correlation was observed between nitrate reductase (NR) activity and the temporal pattern of NO production since both parameters exhibited increased levels in immature fruits. The ripening of theses fruits was accompanied by a transitory reduction in endogenous NO levels whereas its degradation rates were maintained reduced all over the ripening process, thereby suggesting the existence of a more stable NO reservoir such as GSNO or some other S-nitrosothiol. In general light signaling positively influenced both NO production and degradation in mature green tomato fruits. Altogether, the data obtained indicated that tomato NO metabolism is significantly influenced by light, which is able to simultaneous modulate both the production and degradation of this important signaling compound.