Identification and functional characterization of proteins S-nitrosated during salt stress in Klebsormidium nitens

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English. The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations, and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, in order to determine the potential function of these proteins. Among them, we selected an interesting protein called INOSITOL POLYPHOSPHATE MULTIKINASE 2 (IPK2), potentially involved in cell signaling and stress response. We produced it successfully in Escherichia coli and measured its activity in collaboration with the Leibniz-Forschungsinstitut für Molekulare Pharmakologie. The S-nitrosation of IPK2 was proved to occur on Cys125. We generated 2 mutants, IPK2(C125A) and IPK2(C125S), and observed the conservation of their activity and soon will be proceeding to the measurement after S-nitrosation of the wild type and mutated proteins. Structure analysis were also caried in silico with alphafold, giving insights of potential NO effects. The identification and functional analysis of S-nitrosated proteins in K. nitens, this project will provide a better understanding of the functions of NO in unicellular green algae with NOS.