Proteome-wide tagging with an H<sub>2</sub>O<sub>2</sub> biosensor reveals highly localized and dynamic redox microenvironments.

Kritsiligkou, Paraskevi; Bosch, Katharina; Shen, Tzu Keng; Meurer, Matthias; Knop, Michael; Dick, Tobias P · Proc Natl Acad Sci U S A · 2023

basic_science · Level V

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Abstract

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H<sub>2</sub>O<sub>2</sub> can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H<sub>2</sub>O<sub>2</sub> generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H<sub>2</sub>O<sub>2</sub> sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H<sub>2</sub>O<sub>2</sub>-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H<sub>2</sub>O<sub>2</sub> availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H<sub>2</sub>O<sub>2</sub> generation is much more localized and functionally differentiated than previously recognized.

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