Analyse protéomique et biochimique de la nitrosylation et glutathionylation chez l'organisme photosynthétique Chlamydomonas reinhardtii

Acteurs des mécanismes moléculaires de signalisation cellulaire, les espèces réactives de l'oxygène (ROS) et les espèces réactives de l'azote (RNS) agissent comme des molécules signal transférant des informations extracellulaires ou intracellulaires et induisant des réponses spécifiques. Les ROS/RNS agissent principalement via un ensemble de modifications post-traductionnelles réversibles des résidus thiols sur les protéines parmi lesquelles la nitrosylation et la glutathionylation apparaissent comme des éléments jouant un rôle important dans de nombreux processus cellulaires fondamentaux et impliqués dans nombre de maladies humaines. Bien que présents chez les organismes photosynthétiques, ces modifications ont été moins étudiées. Mon projet était d'étudier, in vivo, chez l'algue Chlamydomonas reinhardtii, la dynamique de la nitrosylation et de la glutathionylation, en utilisant une combinaison d'approches multidisciplinaires incluant protéomique, biochimie et biologie moléculaire. En réponse au stress nitrosatif, 492 protéines S-nitrosylées in vivo et 392 sites de nitrosylation ont été identifiés par spectrométrie de masse. Ces protéines participent à un large éventail de processus biologiques tels que la photosynthèse et la réponse au stress. Avec une stratégie similaire, l’analyse de la glutathionylation en réponse à des stresses physiologiques de forte lumière et de choc thermique, a révélé des voies spécifiques de réponse au stress. En parallèle, la dépendance redox des mécanismes moléculaires sous-jacents a été examinée pour la GAPDH cytoplasmique et l’isocitrate lyase, mais aussi la triosephosphate isomérase et la phosphoglycérate kinase chloroplastiques.Actors of the molecular mechanism of cell signaling, reactive oxygen species (ROS) and reactive nitrogen species (RNS) act as signaling molecules to transfer extracellular or intracellular information and elicit specific responses. ROS/RNS mainly act through a set of reversible post-translational modifications of thiol residues on proteins among which nitrosylation and glutathionylation have emerged as key elements playing a major role in numerous fundamental cell processes and implicated in a broad spectrum of human diseases. Despite ROS and RNS are present in photosynthetic organisms, such modifications have been less studied. My project was to investigate in the green algae Chlamydomonas reinhardtii, the in vivo dynamics of nitrosylation and glutathionylation, using a combination of multidisciplinary approaches including proteomic, biochemistry and molecular biology. In response to nitrosative stress, 492 in vivo s-nitrosylated proteins and 392 sites of nitrosylation were identified by mass spectrometry. These proteins were found to participate in a wide range of biological processes and pathway such as photosynthesis, stress response and carbohydrate metabolism. Employing a similar strategy, analysis of glutathionylation in response to physiological stresses, specifically high light and heat stress revealed specific stress dependent targeted pathways. In a second part, the redox dependence of the underlying molecular mechanisms was examined for the cytoplasmic GAPDH and ICL, but also the chloroplastic TPI and PGK. This work has highlighted the existence of a strong interplay between these redox modifications. a complex redox networ

Confrontation de cas autour de la pratique de l'Enchytrismos : de Reims à Toulouse, cohérence et disparité

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Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed

Efficacy and safety of TOXO KO vaccine to prevent ocular toxoplasmosis in congenital murine model

National audienc

A light switch based on protein S-nitrosylation fine-tunes photosynthetic light-harvesting in the microalga Chlamydomonas reinhardtii

International audiencePhotosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform 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 its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant 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. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting

A Light Switch Based on Protein S-Nitrosylation Fine-Tunes Photosynthetic Light Harvesting in Chlamydomonas

Berger H, De Mia M, Morisse S, et al. A Light Switch Based on Protein S-Nitrosylation Fine-Tunes Photosynthetic Light Harvesting in Chlamydomonas. Plant Physiology. 2016;171(2):821-832.Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform 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 its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant 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. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting

Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

International audienceNitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In the present work, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches including activity assays, the biotin switch technique, site-directed mutagenesis and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by transnitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step towards formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GapC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed

Redox regulation of the Calvin\u2013Benson cycle: something old, something new

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin-Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin-Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin-Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresse