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

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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Figure 1: RoGFP2-Tsa2ΔCR is an ultrasensitive H2O2 sensor.
Figure 2: RoGFP2-Tsa2ΔCR is more sensitive than roGFP2-Orp1 or HyPer.
Figure 3: Decamerization with endogenous Tsa1 permits the generation of catalytically inactive sensors.
Figure 4: Basal cytosolic H2O2 levels are tightly coupled to oxygen partial pressure.
Figure 5: Cytosolic and mitochondrial matrix H2O2 levels can vary independently of each other.
Figure 6: Cellular growth rate correlates with basal mitochondrial matrix H2O2 levels.

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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.

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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.

Corresponding authors

Correspondence to Bruce Morgan or Tobias P Dick.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1 – 15 and Supplementary Tables 1 and 2. (PDF 2499 kb)

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Morgan, B., Van Laer, K., Owusu, T. et al. Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. Nat Chem Biol 12, 437–443 (2016). https://doi.org/10.1038/nchembio.2067

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