Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens in response to salt stress
Résumé
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 Snitrosation, 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 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 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.