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RESEARCH PRODUCT

Role of nitric oxide synthases from Klebsormidium nitens: structural characterization and identification of protein partners

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Nitric oxide (NO) is an important cellular signalling molecule regulating various physiological processes, in both animals andplants. In animals, NO synthesis is mainly catalysed by NO synthase (NOS) enzymes. During biotic or abiotic stresses, NOSlike activities that are sensitive to mammalian NOS inhibitors have been detected in plants, although no sequences encodingthe well described mammalian NOS are highlighted in land plants. Interestingly, we identified NOS-like sequences in 20 algaespecies. Among them, NOSs are found in Klebsormidium nitens the model alga to study the early transition step from aquaticalgae to land plants.As mechanisms governing NO synthesis and signalling in green lineage remain unclear, the study of NOSs from K. nitens(KnNOS) through (i) characterization of their nucleotide and protein sequences, (ii) their expression levels, and (iii) theidentification of partners proteins might be an important breakthrough knowledge. To achieve this goal, nucleotide sequenceswere verified by RACE-PCR and sequencing, and after cloning, tagged-proteins were produced. Then the identification ofprotein partners will be performed by pull-down experiments followed by mass spectrometry analysis.Currently, after re-annotating the nucleotide sequence of KnNOSs, a yeast His-tagged protein production strategy has beenadopted. While waiting for the final results of the KnNOSs interactome, an in sillico study was conducted using the BioGriddatabase and human NOS interaction data. This finding opens the way for a deeper characterization of KnNOSs and its proteinpartners and will facilitate further investigation of NO signalling in green lineage.