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Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes

Nature Chemical Biology volume 12, pages 437443 (2016) | Download Citation

Abstract

Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are 50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2ΔCR probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate.

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Acknowledgements

We acknowledge funding from the German Research Foundation (DFG) Priority Program (SPP) 1710 and the DFG Collaborative Research Center (SFB) 1036 to T.P.D., and a DKFZ visiting scientist fellowship to B.M.

Author information

Author notes

    • Bruce Morgan
    •  & Koen Van Laer

    These authors contributed equally to this work.

Affiliations

  1. Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.

    • Bruce Morgan
    • , Koen Van Laer
    • , Theresa N E Owusu
    • , Daria Ezeriņa
    • , Daniel Pastor-Flores
    • , Prince Saforo Amponsah
    • , Anja Tursch
    •  & Tobias P Dick
  2. Cellular Biochemistry, University of Kaiserslautern, Kaiserslautern, Germany.

    • Bruce Morgan

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Contributions

T.P.D., B.M. and K.V.L. conceived the project. B.M., K.V.L. and T.N.E.O. performed most experiments. D.E., D.P.-F., P.S.A. and A.T. contributed to experiments. T.P.D., B.M. and K.V.L. analyzed the data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bruce Morgan or Tobias P Dick.

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1 – 15 and Supplementary Tables 1 and 2.

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DOI

https://doi.org/10.1038/nchembio.2067

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