Mechanisms and Applications of Redox-Sensitive Green Fluorescent Protein-Based Hydrogen Peroxide Probes.

Significance: Genetically encoded hydrogen peroxide (HO) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular HO changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔC offer considerably improved HO sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous HO levels.

Critical Issues: The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain.

Future Directions: The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal HO levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as HO probes, roGFP2-based HO sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.