Proteome-wide tagging with an H₂O₂ biosensor reveals highly localized and dynamic redox microenvironments

Area of Science:

• Cellular Biology
• Biochemistry
• Oxidative Stress

Background:

• Hydrogen peroxide (H2O2) is a key signaling molecule regulating protein function through reversible thiol oxidation.
• The intracellular sources and precise locations of H2O2 generation remain largely unidentified, hindering a full understanding of its signaling pathways.

Purpose of the Study:

• To develop and apply a high-throughput method for identifying endogenous H2O2 generation sites at the protein level in yeast.
• To investigate the dynamic and localized nature of H2O2 production under varying metabolic conditions.

Main Methods:

• Construction of a comprehensive yeast library expressing the H2O2 sensor HyPer7 fused to all protein-coding open reading frames (ORFs).
• Generation of a control library using a redox-insensitive sensor (SypHer7) for background correction.
• Screening of both libraries under diverse metabolic conditions to pinpoint H2O2-generating protein environments.

Main Results:

• Identification of specific proteins and protein complexes as localized H2O2 generation sites.
• Observation of dynamic and condition-specific changes in H2O2 availability around different cellular targets.
• Demonstration that H2O2 production is not uniform but highly compartmentalized within the cell.

Conclusions:

• Intracellular H2O2 generation is significantly more localized and functionally specialized than previously assumed.
• This localized H2O2 signaling provides a new framework for understanding redox regulation of cellular processes.
• The developed screening platform enables future investigations into H2O2 dynamics in various biological contexts.