title: 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.
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.|Cellular Biochemistry, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Bruce
authors.initials: B
authors.lastname: Morgan
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Koen
authors.initials: K
authors.lastname: Van Laer
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Theresa N E
authors.initials: TN
authors.lastname: Owusu
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Daria
authors.initials: D
authors.lastname: Ezeriņa
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pastor-Flores
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Prince Saforo
authors.initials: PS
authors.lastname: Amponsah
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Tursch
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Tobias P
authors.identifier: 0000-0003-1367-973X
authors.initials: TP
authors.lastname: Dick
mesh_terms: D003600:Cytosol / Q000378:metabolism; D006706:Homeostasis; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010313:Partial Pressure; D054464:Peroxiredoxins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D013997:Time Factors
chemical_list: D015335:Molecular Probes; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D010100:Oxygen