Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N2), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium Anabaena PCC 7120. Apart from performing oxygenic photosynthesis, Anabaena PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. NtcA from Anabaena PCC 7120 was heterologously expressed in E. coli and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA in vitro using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N8 – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif. Furthermore, an NtcA binding site was found in the promoter region of the hupSL gene, encoding an uptake hydrogenase in Nostoc punctiforme (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.

La régulation redox est un mécanisme ancien présent chez les organismes biologiques et impliquée dans diverses voies métaboliques. En particulier chez les organismes photosynthétiques elle est responsable des mécanismes d‘adaptation rapide dans un environnement constamment modifié. Dans les chloroplastes le système ferrédoxine/thiorédoxine est la cascade redox principale qui relie l‘activité de plusieurs enzymes plastidiales à la source lumineuse. Le rôle central dans ce système est joué par la ferrédoxine-thiorédoxine réductase (FTR), une protéine hétérodimérique qui récupère des électrons à partir de la ferrédoxine photoréduite et les transfère pour réduire des thiorédoxines plastidiales. Ces protéines peuvent alors réduire des enzymes cibles, requérant l‘accessibilité de paires de cystéines dans un disulfure dont la réduction résulte en une activation/ inactivation de la cible. Jusqu‘à présent des plantes viables n‘ont pu être obtenues en l‘absence de ce système de régulation. Dans cette thèse des secteurs du système redox ont été explorés chez la plante modèle Physcomitrella patens (une mousse). Par manipulation de gènes l‘influence de l‘enzyme FTR sur la croissance et le développement de la plante a été analysée suivant différents paramètres. De manière à impacter la fonction de la réductase des changements nucléotidiques simples ont été introduits au niveau des codons programmant les cystéines catalytiques et dans un deuxième temps le gène complet a été supprimé. De façon inattendue nous n‘avons observé aucun effet significatif sur la viabilité et le développement des plantes mutantes. De plus, nous avons détecté dans P. patens des thiorédoxines additionnelles absentes chez les plantes à graine qui sont fonctionnelles vis à vis des enzymes cibles mais non-réduites par la FTR. Ceci rend possible un scénario de compensation chez les mutants via un système de réduction FTR-indépendant qui reste à caractériser. Deux des cibles photorégulées, la fructose-1,6-bisphosphatase (FBPase) et la sédoheptulose-1,7-bisphosphatase (SBPase), fonctionnent dans la phase de régénération du cycle de Calvin-Benson cycle et elles possèdent plusieurs caractéristiques de catalyse et de régulation similaires. En combinant des approches biochimiques et structurales, une comparaison fonctionnelle et structurale des deux phosphatases de P. patens a été conduite. De plus l‘analyse phylogénétique a révélé une origine procaryotique indépendante des deux séquences en dépit de leurs similitudes structurales et catalytiques. De plus trois articles de revue résument la plasticité et la représentativité du modèle P. patens pour la recherche forestière, les principes généraux de la régulation redox relativement aux aspects évolutifs et fonctionnels chez les plantes ainsi que l‘ état de l‘art de la régulation redox chez les espèces ligneuses en utilisant principalement le peuplier comme modèle / Redox regulation is an ancient mechanism present in biological organisms and is involved in diverse cellular pathways. In particular in photosynthetic organisms it is responsible for fast adaption mechanisms to a constantly changing environment. In chloroplasts the ferredoxin/thioredoxin system represents the main redox regulatory cascade which links the activity of several plastid enzymes to the energy source, light. A central role in this system is played by the heterodimeric ferredoxin-thioredoxin reductase (FTR), which gains electrons from the photo-reduced ferredoxin and transfers those further on via reduction to plastidal thioredoxins. Those proteins in turn reduce their target enzymes and require therefore the availability of redox sensitive cysteine pairs whose reduction results in an inactivation/activation switch of the targets. So far no viable plants could be obtained in complete absence of this redox regulation system. In this thesis single sections of the system were explored in the model plant Physcomitrella patens. Through gene manipulation the influence of the FTR enzyme on plant growth and development was analysed. In order to impact on the function of the reductase, firstly single nucleotide exchange of the catalytic cysteines was performed and later on the gene was completely deleted. Surprisingly, no significant effect could be observed on the viability and development of mutant lines compared to WT plants. Furthermore we found that P. patens possesses in contrast to seed plants additional thioredoxins which are functional for reduction of FTR target enzymes but are most likely not supplied with electrons by this reductase. Thus a possible rescue scenario independent of FTR could be assumed for P. patens and also by other redox regulation systems present in chloroplasts. Two of the FTR target enzymes, fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase, are functional in the regeneration phase of the Calvin-Benson cycle and share similar characteristics in regulation and catalysis. By combining biochemical and structural approaches, a functional comparison of both phosphatases was conducted using cDNAs from P. patens. A stricter TRX-dependent regulation and catalytic cleavage ability for both substrates, FBP and SBP, could be observed for PpSBPase, whereas PpFBPase is only capable of cleaving FBP. By obtaining the oxidized X-ray structure of both enzymes these observations can be associated with the distinct positions of regulatory sites and the various sizes of the substrate binding pocket. In addition, the phylogenetic analysis revealed an independent prokaryotic origin for both phosphatases. Furthermore we summarized in three review articles the amenability of P. patens as model plant for forest research, the general principles of redox regulation in respect of evolution and functional mechanisms in plants, and the current state of the art in forest redox regulation using poplar as exemplary model

La phosphoribulokinase (PRK) et la glycéraldéhyde 3-phosphate déshydrogénase (GAPDH) sont deux enzymes-clés du cycle de Calvin-Benson. Leurs activités sont régulées par l’intermédiaire de la CP12, une protéine intrinsèquement désordonnée. Au cours de la transition lumière-obscurité, la GAPDH, la CP12 et la PRK forment un complexe supramoléculaire au sein duquel l’activité des enzymes est inhibée. Dans les travaux présentés ici, nous nous sommes intéressés à la formation de ce complexe et à la dynamique de ses composants. Nous avons montré pour la première fois que les résidus cystéine Cys243 et Cys249 de la PRK sont essentiels à la formation du complexe GAPDH-CP12-PRK et qu’ils peuvent former un pont disulfure en présence de CP12. Nous avons également étudié la dynamique de la CP12 en présence de ses partenaires, et observé que la CP12 adopte une conformation beaucoup plus compacte en présence de GAPDH et de PRK. La glutathionylation (formation d’un pont disulfure mixte entre une molécule de glutathion et un résidu cystéine appartenant à une protéine) est une modification post-traductionnelle associée au stress oxydant qui affecte dix enzymes du cycle de Calvin-Benson, y compris la GAPDH et la PRK. Nous avons étudié l’impact de la glutathionylation sur ces enzymes, et montré que l’inactivation de la PRK naît de l’encombrement du site de fixation de l’ATP.Enfin, la dernière partie de ces travaux est centrée sur l’adénylate kinase 3 de C. reinhardtii, une enzyme impliquée dans le métabolisme de l’ATP et qui possède une extension similaire à la CP12. Cette première étude montre que cette extension augmente la stabilité de l’ADK 3 et intervient dans sa glutathionylation. / Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are two key enzymes of the Calvin-Benson and their activities are redox-regulated through the intervention of CP12, a intrinsically disordered protein. During the light-to-dark transitions, GAPDH, CP12 and PRK form a supramolecular complex in which the enzymes are strongly inhibited; this complex is dissociated during the dark-to-light transition and the active enzymes are released.In the work presented here, we studied the formation of the complex and the dynamics of its components. For the first time, we showed that two cysteine residues of PRK, Cys243 and Cys249, are essential to the assembly of the GAPDH-CP12-PRK complex, and can form a disulfide bridge in presence of CP12.Glutathionylation (the formation of a mixed disulfide bridge linking one glutathione molecule and a cysteine residue from a protein) is a post-translational modification associated with oxidative stress that affects ten of the Calvin-Benson enzymes, including GAPDH and PRK, and we show that the inactivation of PRK by glutathionylation is caused by the blockage of the ATP binding site by glutathione.The last part of this work is centered around adenylate kinase 3 from C. reinhardtii, an enzyme tied to the energetic metabolism of the cells that presents a CP12-like C-terminal extension. Our results suggest that this CP12-like “tail” improve the stability of ADK 3 and participates in tis glutathionylation.

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.

No description available.

L’équilibre rédox entre les niveaux des espèces réactives de l’oxygène et de l’azote (ROS, RNS) et les espèces antioxydantes cellulaires est déterminant pour le fonctionnement normal de la cellule et sa viabilité. Le déséquilibre redox ou « stress oxydant » peut altérer les voies de signalisation cellulaires et générer des dommages sur les protéines, les lipides et l’ADN des cellules. Il est ainsi associé à de nombreuses pathologies, notamment les cancers. Les cellules cancéreuses présentent une dérégulation redox importante et un stress oxydant basal intrinsèque plus élevé par rapport aux cellules normales. Elles sont donc très dépendantes des systèmes antioxydants pour leur viabilité. Ainsi, l’administration de drogues qui i) génèrent des ROS / RNS additionnelles ou ii) inhibent les systèmes antioxydant cellulaires, permet une cytotoxicité sélective contre les cellules cancéreuses. C’est la base biologique de la « thérapie anticancéreuse basée sur la modulation de l’équilibre redox». Dans ce contexte, nos travaux ont pour but de décrypter les mécanismes redox derrière l’activité anticancéreuse de la vitamine C (VitC) et de l’auranofin (AUF), seuls ou en combinaison, dans le modèle du cancer du sein. La VitC à des concentrations pharmacologiques élevées présente des propriétés pro-oxydantes. Dans cette étude, l’activité anticancéreuse de la VitC contre les lignées du cancer du sein est associée à une génération extracellulaire et intracellulaire de peroxyde d'hydrogène (H₂O₂) accompagnée d'une oxydation intracellulaire du glutathion (GSH). L’approche protéomique «redoxome» a révélé que la VitC induit une altération de l'état rédox d’enzymes antioxydantes clés et d'un certain nombre de protéines contenant des cystéines, impliquées dans les métabolismes de l’ARN et l’ADN et dans les processus énergétiques. La VitC est également responsable d’un retard dans la progression du cycle cellulaire et d’une inhibition de la traduction. Finalement, des analyses bioinformatiques ont montré que les niveaux d'expression de la peroxiredoxine 1 (PRDX1) sont corrélés à la cytotoxicité différentielle de la VitC dans les cellules cancéreuses du sein. L'AUF, un antirhumatismal, est un inhibiteur des thiorédoxines réductases qui a reçu une attention croissante pour son activité anticancéreuse. Nos travaux montrent que l’AUF inhibe également le système antioxydant du GSH et que cette inhibition est primordiale pour son activité anticancéreuse. L’AUF modifie l'état redox de nombreuses protéines impliquées dans la prolifération et le cycle cellulaire, et provoque une déplétion des dNTPs et un arrêt du cycle cellulaire. De façon remarquable, nous avons démontré que la combinaison de l’AUF et de la VitC présente une cytotoxicité accrue, synergique, médiée par H₂O₂ dans les cellules MDA-MB-231 et d'autres lignées cellulaires du cancer du sein sans trop affecter les cellules normales. In vivo, l’efficacité de la combinaison AUF/VitC a été validée sur des xénogreffes de MDA-MB-231 chez les souris sans présenter une toxicité notable, tandis que l'administration de l’AUF ou de la VitC en monothérapie n’inhibe pas la croissance tumorale. Enfin, les analyses protéomiques, bioinformatiques et fonctionnelles ont identifié la prostaglandine réductase 1 (PTGR1) comme biomarqueur prédictif de la réponse des cellules cancéreuses du sein à la combinaison AUF/VitC. En résumé, ces résultats contribuent à une meilleure compréhension des mécanismes anticancéreux de la VitC et de l'AUF, seuls et en combinaison. En particulier, la combinaison de ces deux médicaments disponibles et non toxiques pourrait être efficace contre le cancer du sein triple négatif et potentiellement d'autres cancers présentant des propriétés redox similaires. Ainsi, une évaluation préclinique et clinique de ces traitements ouvrira la voie à des nouvelles thérapies anticancéreuses basées sur la modulation de l’équilibre redox cellulaire. / Reactive oxygen and nitrogen species (ROS, RNS) homeostasis and intracellular reductive/oxidative (redox) dynamics play a key role in regulating cell fate and are critical for normal cellular functions. Oxidative stress via the disruption of redox homeostasis can lead to aberrant cell signaling and toxic oxidative damage of DNA, lipids and proteins, and is therefore associated with human pathologies such as cancers. Cancer cells experience extensive redox deregulation and generally exhibit higher intrinsic basal oxidative stress than normal cells, as a consequence, they are more dependent on their antioxidant systems for survival. Thus, the administration of a drug generating additional ROS / RNS or inhibiting cellular antioxidant systems will exert a selective cytotoxicity towards cancer cells while sparing their normal counterparts. This is the biological basis for « redox-based anticancer therapy ». The work described here aims to investigate the redox-based anticancer activity of vitamin C (VitC) and auranofin (AUF), as single drugs or in combination, in breast cancer model. VitC at high pharmacological concentrations shows pro-oxidant properties. In this study, we showed that VitC anticancer activity against breast cancer cell lines was associated to extracellular and intracellular generation of hydrogen peroxide (H₂O₂), accompanied by the oxidation of intracellular glutathione (GSH). A “redoxome” proteomics approach revealed that VitC induces alterations of the redox state of key antioxidant enzymes and a number of cysteines-containing proteins including many proteins involved in RNA and DNA metabolisms and energetic processes. Cell cycle arrest and translation inhibition are associated with VitC-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlate with VitC differential cytotoxicity in breast cancer cells. AUF, an antirheumatic drug and known inhibitor of thioredoxin reductases, has been repurposed recently as a potent anticancer drug. We showed that AUF acts on both the thioredoxin and GSH systems and its impact on GSH system is essential for its anticancer activity. AUF alters the redox state of a number of nucleic acid-binding proteins involved in cell proliferation, cell division and cell cycle, triggering dNTP depletion and cell cycle arrest. Importantly, we observed that the combination of AUF and VitC reveals a synergetic and H₂O₂-mediated cytotoxicity towards MDA-MB-231 cells and other breast cancer cell lines without much impact on normal cells, thus decreasing the cytotoxic concentrations of AUF or VitC single drug. The anticancer potential of AUF/VitC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects, while administration of AUF or VitC as a single agent failed to suppress tumor growth. Finally, SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell response to AUF/VitC combination, thus identifying a potential predictive biomarker. Overall, these results provide new insights into the anticancer mechanisms of VitC and AUF, as single drugs and in combination. In particular, this combination of two non-toxic and commonly available drugs could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties. Further assessment in preclinical and clinical studies of these drugs and combinations could open new avenues for redox-based anticancer therapy.

<p>The Muoio lab studies the interplay between lipid whole body energy balance,</p><p>mitochondrial function and insulin action in skeletal muscle. Data from our lab suggests that lipid-induced insulin resistance in skeletal muscle may stem from excessive incomplete oxidation of fatty acids, which occurs when high rates of &beta;-­oxidation exceed TCA cycle flux (Koves et al., 2005; Koves et al., 2008). Most notably, we have shown that mice with a genetically engineered decrease in mitochondrial uptake and oxidation of fatty acids are protected against diet-­induced insulin resistance (Koves et al., 2008). This</p><p>suggests that an excessive and/or inappropriate metabolic burden on muscle</p><p>mitochondria provokes insulin resistance. Our working model predicts that: 1) high rates of incomplete &beta;-oxidation reflect a state of &rdquo;mitochondrial stress,&rdquo; and 2) that energy-overloaded mitochondria generate a yet unidentified signal that mediates insulin</p><p>resistance. One possibility is that this putative mitochondrial-derived signal stems from redox imbalance and disruptions in redox sensitive signaling cascades. Therefore, we are interested in identifying molecules that link redox balance, mitochondrial function and insulin action in skeletal muscle. The work described herein identifies thioredoxin-interacting protein (TXNIP) as an attractive candidate that regulates both glucose homeostasis and mitochondrial fuel selection.</p><p>TXNIP is a redox sensitive, &alpha;-arrestin protein that has been implicated as a negative regulator of glucose control. Mounting evidence suggested that TXNIP might play a key role in regulating mitochondrial function; however, the molecular nature of this relationship was poorly defined. Previous studies in TXNIP knockout mice reported that deficiency of this protein compromises oxidative metabolism, increases glycolytic activity and promotes production of reactive oxygen species (ROS), while also affording protection against insulin resistance. Therefore, we hypothesized that TXNIP might serve as a nutrient sensor that couples cellular redox status to the adjustments in mitochondrial function. We tested this hypothesis by exploiting loss of function models to evaluate the effects of TXNIP deficiency on mitochondrial metabolism and respiratory function.</p><p>In chapter 3, we comprehensively evaluated oxidative metabolism, substrate</p><p>selection, respiratory kinetics and redox balance in mice with total body and skeletal muscle-­specific TXNIP deficiency. Targeted metabolomics, comprehensive bioenergetics analysis, whole-body respirometry and conventional biochemistry showed that TXNIP deficiency results in reduced exercise tolerance with marked impairments in skeletal muscle oxidative metabolism. The deficits in substrate oxidation were not secondary to decreased mitochondrial mass or increased H₂O₂ emitting potential from the electron transport chain. Instead, the activities of several mitochondrial dehydrogenases involved in branched-chain amino acid and ketone catabolism, the tricarboxylic acid (TCA) cycle and fatty acid &beta;-oxidation were significantly diminished in TXNIP null muscles. These deficits in mitochondrial enzyme activities were accompanied by decreased protein abundance without changes in mRNA expression. Taken together, these results suggest that in skeletal muscle TXNIP plays an essential role in maintaining protein synthesis and/or stability of a subset of mitochondrial dehydrogenase enzymes that permit muscle use of alternate fuels under conditions of glucose deprivation.</p><p>Based on these conclusions, we questioned whether additional regulatory</p><p>mechanisms could contribute to the reduced oxidative metabolism in the absence ofTXNIP. Several metabolic enzymes of the TCA cycle have been shown to be redox-sensitive protein targets regulated by the thioredoxin (TRX1/TRX2) and glutathione (GSH) redox-mediated circuits. TXNIP has been shown to respond to oxidative stress by shuttling to the mitochondria where it binds to TRX2 and/or other proteins, thus affecting downstream signaling pathways, such as the apoptotic cascade. Therefore, we speculated whether there was a role for redox imbalance in mediating the mitochondrial phenotype of the TXNIP knockout (TKO) mice. In chapter 4, we present preliminary evidence that increased glucose uptake promotes non-mitochondrial ROS production, causing a shift in redox balance, decreased GSH/GSSG, and S-glutathionylation of &alpha;-­ketoglutarate dehydrogenase (&alpha-KGD). This post-translational modification protects the protein from permanent oxidative damage, but at the cost of reversible loss of activity and subsequent disruption of TCA cycle flux that contributes, in part, to the diminished oxidative metabolism observed in the TXNIP deficient mice.</p><p>In aggregate, this work sheds new light onto the physiological role of TXNIP in</p><p>skeletal muscle as it pertains to substrate metabolism and fuel switching in response to nutrient availability. This work has important implications for metabolic diseases such as obesity and type 2 diabetes, which are characterized by marked disruptions in fuel selection.</p> / Dissertation

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<p>Balans redoks potencijala u živoj ćeliji predstavlja imperativ održavanja zdravog fenotipa, i u&nbsp;krajnjem, njenog preživljavanja. Nitrozativni stres može ozbiljno naru&scaron;iti ćelijsku redoks&nbsp;homeostazu i, u kombinaciji sa oksidativnim stresom, uticati na ćelijsku proliferaciju i&nbsp;diferencijaciju, a u nekim slučajevima i na aktivaciju maligne transformacije&nbsp;U ovom radu ispitivani su efekti donora NO natrijum-nitroprusida na dve ćelijske linije u&nbsp;kulturi: transformisane&nbsp; ćelije mi&scaron;ijih fibroblasta (L929) i maligne&nbsp; ćelije humane&nbsp;eritroleukemije (K562). Natrijum- nitroprusid (SNP) je fotoreativan molekul sa veoma&nbsp;kratkim poluživotom koji izaziva koncentraciono - zavisnu proliferaciju ili inhibiciju<br />ćelijskog rasta in vitro.NO izaziva različite efekte u zavisnosti od eksperimentalnog modela,&nbsp;svoje relativne koncentracije kao i okruženja u kojem nastaje. Ispitivanja mogućnosti direktne&nbsp;transformacije azot oksida u redoks aktivne vrste kao &scaron;to su nitrozonijum katjon (NO⁺) i&nbsp;nitroksil anjon (NO^-/HNO) i direktni efekti tih redoks potomaka u ćeliji tek su u začetku. U&nbsp;na&scaron;im eksperimentima, kori&scaron;ćenjem donora NO - natrijum nitroprusida (SNP) i dve vrste&nbsp;superoksid dismutaza, CuZn-SOD i Mn-SOD, stvorili smo uslove generisanja vi&scaron;e vrsta&nbsp;signalnih molekula i ispitali odgovor transfomisanih (L929) i malignih (K562) ćelija na njih.&nbsp;Rezultati eksperimenata pokazuju da izabrani parametri (količina slobodnih tiolnih grupa i&nbsp;glutationa) mogu biti relevantni za praćenje efekata egzogenog azot oksida i njegovih redoks&nbsp;potomaka kod različitih, transformisanih i malignih ćelijskih linija.</p> / <p>The redox potential balance in the living cell isthe imperative of continuation of healthy phenotype, and subsequently of its survival. Nitrosative stress may seriously damage cell&#39;s redox homeostasis, and in combination with oxidative stress may influence cell proliferation and differentiation, in some cases even activation of malignant transformation. This paper investigates effects of sodium nitroprusside as&nbsp; NO donor on two cell lines in culture: transformed cells of mice fibroblasts (L929) and malignant cells of human eritroleukemia (K562). The sodium nitroprusside(SNP)&nbsp; is a photo reactive molecule with very short half-life, causing concentration- dependant proliferation or inhibition of cell growth in vitro.The NO causes different effects depending on experimental model, its relative concentration and environment&nbsp; where it is formed. Investigations of possibility of direct transformation from nitrogen oxide to redox-active species as nitrosonium cation (NO+) and nitroxyl anion (NO &minus;/HNO), as well as direct effects ofthose redox descendants within the cell are only in beginning. In our experiments,by using sodium nitroprusside (SNP) as NO donor and&nbsp; two kind of superoxide dismutase, CuZn-SOD and Mn-SOD, we created conditions to generate several kinds of signal molecules and investigated reaction of transformed (L929) and malignant (K562) cells tothose. Results of experiments are showing the parameters chosen (amount of free thiol groups and glutathione) may be relevant in measuring the effect of exogenous nitrate oxideand its redox descendants in different,&nbsp; both transformed and malignant cell lines.</p>

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science