CE44 - Biochimie du Vivant

Dissecting ER redox metabolism in ER physiology – ERRed2

Submission summary

During endoplasmic reticulum (ER) stress, a complex interplay opposes the ER thiol oxidation and reduction pathways, with concurrent cellular production of H2O2, which together with UPR (Unfolded Protein Response) signaling, dictate the ability of the cell to adapt to ER stress or to undergo cell death. Despite its high relevance to human pathology, this redox interplay remains largely ununderstood. To address this question, we propose to elucidate two of the major conundrum of ER redox metabolism: (i) whether and how the thioredoxin and glutathione (GSH) thiol-reducing pathways contribute to oxidative protein folding as sources of reducing equivalents, and (ii) what are the sources and functions of the H2O2 produced during ER stress. Our strategy is to establish tools to specifically manipulate these pathways, using both yeast and mammalian cellular models that largely share similar ER oxidative protein folding machineries, the former for its mechanistic simplicity and experimental power and the latter for its relevance to human pathology. Having hands on these pathways will best enable dissecting their role. This strategy is organized into five work packages (WP). (WP1) We established a mammalian GSH ER transport assay to show that, as in yeast, GSH is imported into the ER along a concentration gradient imposed by its synthesis in the cytosol and its consumption in the ER by oxidation by Ero1-PDI. We will use this assay to identify the GSH ER transport system, in both a chemical and a siRNA-based pangenomic high throughput (HT) screens, each as backup of the other. (WP2) Cytosolic thioredoxins serve as ER reducing power but are absent in the ER, calling for the presence of an ER transmembrane (TM) thiol-disulfide relay system that links them to ER luminal reductases. We will search for this TM system by identification of the yeast thioredoxin repertoire using both a HT genetic screen based on the DHFR protein complementation assay conducted by our Israeli partner, and the pulldown of thioredoxin substrates by virtue of disulfide attachment. Mammalian homologues of the yeast TM protein and LMF1, a putative ER TM redox-transfer protein will be characterized. (WP3) The source(s) of the H2O2 produced during ER stress remain controversial by lack of adequate ER detection system. We will establish two innovative methods to detect ER luminal H2O2. The first is based on the horseradish peroxidase APEX2biotin-phenol (BP). The second relies on snapshots of the extent of ER protein S-sulfenylation, after in situ labeling with the alkyne dimedone-derivative DYn-2. Proof-of-principle has been obtained. (WP4) The redox state of Cys residues is the biochemical endpoint that integrate the function(s) and activity of ER thiol-oxidizing and reducing pathways. We will use the OcSILAC procedure established by P2 and P1, a site-centric shotgun MS strategy that simultaneously monitor reversible Cys residues oxidation and protein levels to identify the ER thiol redoxome, which will uniquely map ER thiol-reducing pathways by their thiol-redox fingerprints. OcSILAC will be complemented by global assays of ER proteins S-glutathionylation, which uses clickable GSH, and S-sulfenylation. (WP5) Tools and knowledge obtained in the four other WPs will be applied to tunable yeast and mammalian ER stress physiological systems departing from pharmacological ER stressors, established by our Italian partner, to describe the nature and timing of the redox events occurring during adaptive and lethal ER stress, and to challenge the ER role of thiol-reducing pathways and of H2O2 production. The project should provide a unique model of ER redox metabolism, depicting its impact on ER stress outcome and developing new tools, all relevant to human physiopathology.

Project coordination

Agnès DELAUNAY-MOISAN (Institut des sciences du vivant FRÉDÉRIC-JOLIOT)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

JOLIOT Institut des sciences du vivant FRÉDÉRIC-JOLIOT
JOLIOT Institut des sciences du vivant FRÉDÉRIC-JOLIOT
SMBP Spectrométrie de Masse Biologique et Protéomique

Help of the ANR 615,302 euros
Beginning and duration of the scientific project: September 2019 - 48 Months

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