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Identification and functional characterization of S-nitrosated proteins from the alga Klebsormidium nitens
No full textLe monoxyde d'azote (NO) est un composant ubiquitaire majeur de la signalisation cellulaire, exerçant ses effets en partie par la S-nitrosation des protéines. Cette modification post-traductionnelle peut impacter l’activité des protéines, leur localisation subcellulaire et leur capacité à former des complexes protéiques. Par conséquent, la caractérisation des protéines S-nitrosées est d'un grand intérêt pour élucider les fonctions du NO. Lors d’une précédente étude, notre laboratoire a fourni des preuves que les plantes terrestres ne possèdent pas de NO Synthase (NOS), l'enzyme principale de synthèse du NO chez les métazoaires (Jeandroz et al., 2016). En revanche, quelques espèces d'algues possèdent des protéines apparentées aux NOS, ce qui soulève des questions sur leur biochimie et leurs rôles dans ces organismes. Nous nous sommes concentrés sur l'identification des protéines S-nitrosées chez Klebsormidium nitens, une espèce d'algue d'eau douce possédant deux séquences apparentées à des NOS dans son génome (Chatelain et al., 2022) et considérée comme un modèle pour étudier l'adaptation des plantes à la vie terrestre (Hori et al., 2014). Nous avons identifié 43 protéines candidates présentant des niveaux significativement plus élevés de S-nitrosation en condition de stress salin. Une analyse d'orthologie a été effectuée avec Arabidopsis thaliana pour déterminer la fonction potentielle de ces protéines. Nous avons sélectionné 3 protéines parmi elles, afin d’étudier le rôle de leur S-nitrosation dans la réponse au stress salin. Ces protéines sont la Protéine Phosphatase 1 (PPH1), l’Histone Désacétylase 14 (HDA14) et l'Inositol Polyphosphate Multikinase 2 (IPK2).PPH1 est une protéine cruciale dans l’adaptation des plantes terrestres aux conditions lumineuses. La caractérisation de KnPPH1 a été entamée mais des difficultés sont rapidement survenues, notamment due à une mauvaise annotation de sa séquence dans les banques de données et une difficulté à la produire en système hétérologue. La protéine HDA14 d’A. thaliana et du riz est une histone désacétylase chloroplastique, responsable de la désacétylation de protéines impliquées dans la photosynthèse, ainsi que de la désacétylation de la N-acétyl-sérotonine en sérotonine, des précurseurs à la synthèse de mélatonine. La séquence de KnHDA14 a été vérifiée, ainsi que sa localisation chloroplastique. Nous avons pu la produire en système bactérien et avons confirmé son activité de désacétylase, laquelle semblerait inhibée par le NO. Enfin, chez A. thaliana, IPK2 est une kinase d’inositol phosphates (InsP), des molécules plus ou moins phosphorylées impliquées dans la signalisation cellulaire et la réponse aux stress. Nous avons prouvé que KnIPK2 est capable de phosphoryler, mais aussi, de manière plus surprenante, de déphosphoryler certains InsP. Confirmant nos prédictions in silico, la S-nitrosation in vitro de KnIPK2 par du NO exogène semble inhiber son activité.En résumé, cette thèse a apporté des résultats pionniers sur la signalisation par le NO chez les algues. De plus, ce travail démontre l’implication du NO dans de nouvelles voies de signalisation, et suggère des pistes quant à l’étude de la résistance au stress salin.Nitric oxide (NO) is a major ubiquitous component of cell signaling, exerting its effects partly through protein S-nitrosation. This post-translational modification can impact protein activity, subcellular localization, and the ability to form protein complexes. Therefore, the characterization of S-nitrosated proteins is of great interest for elucidating NO functions. Previously, our laboratory provided provided evidence that land plants do not possess NO Synthase (NOS), the main NO-synthesizing enzyme in metazoans (Jeandroz et al., 2016). However, some algae species possess NOS-relative sequences, raising questions about their biochemistry and roles in these organisms. We focused on identifying S-nitrosated proteins in Klebsormidium nitens, a freshwater algal species which possess two NOS-relative sequences in its genome (Chatelain et al., 2022) and considered a model for studying plant adaptation to terrestrial life (Hori et al., 2014). We identified 43 candidate proteins with significantly higher levels of S-nitrosation under salt stress conditions. Orthology analysis was performed with Arabidopsis thaliana to determine the potential function of these proteins. We selected 3 proteins among them to study the role of their S-nitrosation in the salt stress response. These proteins are Protein Phosphatase 1 (PPH1), Histone Deacetylase 14 (HDA14), and Inositol Polyphosphate Multikinase 2 (IPK2).PPH1 is a crucial protein in terrestrial plant adaptation to light conditions. Characterization of KnPPH1 was initiated, but difficulties quickly arose, mainly due to poor annotation of its sequence in databases and difficulty in producing it in a heterologous system. HDA14 from A. thaliana and rice is a chloroplastic histone deacetylase, responsible for the deacetylation of proteins involved in photosynthesis, as well as the deacetylation of N-acetyl-serotonin into serotonin, precursors to melatonin synthesis. The sequence of KnHDA14 was verified, as well as its chloroplastic localization. We were able to produce it in a bacterial system and confirmed its deacetylase activity, which appears to be inhibited by NO. Finally, IPK2 in A. thaliana is an inositol phosphate (InsP) kinase. InsPs are more or less phosphorylated molecules involved in cell signaling and stress response. We have shown that KnIPK2 can phosphorylate but also, more surprisingly, can dephosphorylate certain InsPs. Confirming our in silico predictions, in vitro S-nitrosation of KnIPK2 by exogenous NO appears to inhibit its activity.In summary, this thesis has provided pioneering results on NO signaling in an algae. Additionally, this work demonstrates the involvement of NO in new signaling pathways, and suggests new ideas for studying salt stress resistance
Les femmes, les sciences et l’Europe. Intervention à la conférence-débat
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Promotion des sciences et plus particulièrement des « femmes en sciences » auprès des élèves de 4ème
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Présentation du métier de chercheur dans le cadre de "Les filles et la Science"
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Etude du rôle du monoxyde d'azote (NO) dans la réponse d'Arabidopsis thaliana au cadmium (liens avec l'homéostasie du fer)
No full textCe travail est une contribution à l'étude du mode d'action du monoxyde d'azote (NO) dans la réponse des plantes aux stress (a)biotiques. Nous avons analysé le rôle du NO dans la toxicité du cadmium (Cd) chez Arabidopsis thaliana. Les plantes ayant absorbé du Cd par les racines produisent du NO dans les racines et les feuilles. Le NO participe à l'inhibition de la croissance des racines induite par le Cd, favorise son absorption et contribue à la réduction de la concentration de calcium (Ca). La production de NO serait la conséquence de la réduction de la concentration en fer. Le NO agirait ensuite comme un signal de carence en fer et activerait l'expression de gènes associés au transport du fer, facilitant ainsi l import du Cd. Le NO pourrait aussi promouvoir l activation de canaux/transporteurs de Ca perméables au Cd et/ou favoriser une compétition entre Cd et Ca. En conclusion, le NO est un médiateur de la toxicité du Cd. Parmi les gènes cibles du NO figure AtNAS4 codant une enzyme catalysant potentiellement la synthèse de nicotianamine (NA). Nous avons caractérisé un mutant d insertion de ce gène qui arbore un taux de NA réduit, indiquant qu AtNAS4 est une source de NA dans les feuilles. L étude du mutant a révélé qu AtNAS4 joue un rôle clé dans l homéostasie du fer, dans la résistance au Cd et en réponse à l infection par Erwinia chrysanthemi. Enfin, nous avons développé une méthode électrochimique de dosage du NO dans le milieu extracellulaire de suspensions cellulaires de tabac. Cette méthode quantitative qui s est avérée sélective et sensible a permis de confirmer la capacité de la cryptogéine, un éliciteur des réactions de défense, à déclencher une production de NO.Here, we investigated the role of nitric oxide (NO) in plant responses to (a)biotic stresses. In a first part, we analyzed the synthesis and role of NO in plants exposed to cadmium (Cd), a non essential and toxic metal. We demonstrated that Cd induces NO synthesis in roots and leaves of Arabidopsis thaliana treated at the root level. NO contributes to Cd-triggered inhibition of root growth and to Cd uptake and is also responsible for Cd-induced inhibition of root Ca accumulation. Taken together, our results suggest that NO contributes to Cd toxicity by favouring Cd versus Ca uptake and by initiating a cellular pathway resembling those activated upon iron deprivation via the activation of expression of genes related to iron uptake. One of the NO-modulated genes in response to Cd treatment is NAS4 (nicotianamine synthase 4) encoding an enzyme potentially catalysing the formation of nicotianamine (NA), a metal chelator. We showed that atnas4, a mutant carrying a T-DNA insertion in AtNAS4, displays a reduced NA content in leaves, indicating that AtNAS4 is a major enzymatic source for NA. This mutant exhibits a hypersensitivity to iron deficiency, to Cd treatment and to infection by the bacterial pathogen Erwinia chrysanthemi. We conclude that AtNAS4 plays a central role in iron homeostasis as well as in Cd and E. chrysanthemi resistance in A. thaliana. The second part of this study dealt with an electrochemical method we developed in order to probe free NO in the extracellular medium of tobacco suspension cells in response to cryptogein, an elicitor of defense responses. This technique provided real-time evidence that cryptogein is able to induce an increase of extracellular NO.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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