| Popis: |
The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitouscomponent of cell signalling. A key signalling mechanism mediating NO effects is Snitrosation, a post-translational modification by which NO can impact the target proteinactivities, subcellular localizations, and capacities to form protein complexes. The identificationof 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 onthe identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, afreshwater algal species possessing a NOS and established as a model to study plants adaptationto land. We applied the Biotin Switch method followed by mass spectrometry analysis. Thismethod allows the purification of S-nitrosated proteins. We found 43 proteins with significantlyhigher S-nitrosation levels in salt response condition. Orthology analysis were performedagainst the model plant Arabidopsis thaliana, in order to determine the potential function ofthese proteins. Among them, we selected an interesting protein called INOSITOLPOLYPHOSPHATE MULTIKINASE 2 (IPK2), potentially involved in cell signaling andstress response. We produced it successfully in Escherichia coli and measured its activity incollaboration with the Leibniz-Forschungsinstitut für Molekulare Pharmakologie. The Snitrosation of IPK2 was proved to occur on Cys125. We generated 2 mutants, IPK2(C125A)and IPK2(C125S), and will observe the conservation of their activity before proceeding to themeasurement after S-nitrosation of the wild type and mutated proteins. Structure analysis werealso caried in silico with alphafold, giving insights of potential NO effects. The identificationand functional analysis of S-nitrosated proteins in K. nitens, this project will provide a betterunderstanding of the functions of NO in unicellular green algae with NOS. |
