Quantitative and temporal REDox PROteomic PROfiling in unicellular eucaryotes – REDPRO2

The aim of this project is to investigate, using an innovative proteomic technology, the in vivo dynamics of nitrosylation and glutathionylation, two major redox modifications involved in cell signaling. The method will allow quantitative and time-resolved detection of both modifications that will be analyzed simultaneously and specifically from each sample. This technology will be combined to bioinformatics modeling to unravel a basic framework of the redox network associated with the responses to diverse physiological conditions or with different genetic backgrounds in two model unicellular eukaryotes: the green alga Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae, including a yeast model of Friedreich's ataxia, a neurodegenerative disease associated with a modification of the cellular redox status. By unraveling major aspects of the dynamics of the redox network, this project is expected to profoundly modify our knowledge of cell signaling mechanisms and thereby have a high impact both for fundamental and biomedical research.

Our analyses allowed to unravel a complex redox network involving more than 1000 proteins regulated by thioredoxins, by nitrosylation or by glutathionylation in the green alga C. reinhardtii and the yeast S. cerevisiae. Targeted biochemical and structural studies allowed to confirm the regulation of several proteins and to determine the molecular mechanisms underlying thioredoxin-dependent redox regulation of autophagy in the alga and the yeast and associated with adaptation of photosynthesis (light harvesting, carbon fixation) to environmental fluctuations in Chlamydomonas.

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The project allowed publications of 9 articles in very good scientific journals and lead to 3 invited conferences including two in major international meetings with more than 1000 partcipants (Brazil, Italy). Several publications are submitted or under preparation.

Living cells have evolved a complex network of signaling pathways and adaptative responses which enable them to survive and adapt to diverse environmental challenges. Our increasing understanding of the molecular mechanism of cell signaling has revealed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) act as signaling molecules to transfer extracellular or intracellular information and elicit specific responses. ROS/RNS mainly act through a set of reversible post-translational modifications of thiol residues on proteins among which nitrosylation and glutathionylation have emerged as key elements playing a major role in numerous fundamental cell processes and implicated in a broad spectrum of human diseases. Emerging evidence suggests the existence of an intricate network of redox signaling pathways with a complex crosstalk between the different types of redox modifications. However, the possible crosstalk between nitrosylation and glutathionylation remains largely unexplored. This is mainly due to the fact that both modifications are almost always studied independently by different research groups rather than simultaneously. We believe that unraveling this crosstalk and analyzing the underlying molecular mechanisms is crucial to get a more complete and accurate understanding of the intricate network of redox regulatory pathways and mechanisms operating in cell regulation and signaling.

The aim of this project is to investigate, using an innovative proteomic technology, the in vivo dynamics of nitrosylation and glutathionylation, two major redox modifications involved in cell signaling. The method will allow quantitative and time-resolved detection of both modifications that will be analyzed simultaneously and specifically from each sample. This technology will be combined to bioinformatics modeling to unravel a basic framework of the redox network associated with the responses to diverse physiological conditions or with different genetic backgrounds in two model unicellular eukaryotes: the green alga Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae, including a yeast model of Friedreich's ataxia, a neurodegenerative disease associated with a modification of the cellular redox status. By unraveling major aspects of the dynamics of the redox network, this project is expected to profoundly modify our knowledge of cell signaling mechanisms and thereby have a high impact both for fundamental and biomedical research.