TY - JOUR
T1 - Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes
AU - Morgan, Bruce
AU - Van Laer, Koen
AU - Owusu, Theresa N E
AU - Ezeriņa, Daria
AU - Pastor-Flores, Daniel
AU - Amponsah, Prince Saforo
AU - Tursch, Anja
AU - Dick, Tobias P
PY - 2016/6
Y1 - 2016/6
N2 - 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.
AB - 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.
KW - Cytosol/metabolism
KW - Homeostasis
KW - Hydrogen Peroxide/analysis
KW - Mitochondria/metabolism
KW - Molecular Probes/metabolism
KW - Oxygen/metabolism
KW - Partial Pressure
KW - Peroxiredoxins/metabolism
KW - Saccharomyces cerevisiae/cytology
KW - Time Factors
U2 - 10.1038/nchembio.2067
DO - 10.1038/nchembio.2067
M3 - Article
C2 - 27089028
VL - 12
SP - 437
EP - 443
JO - Nature Chemical Biology
JF - Nature Chemical Biology
SN - 1552-4450
IS - 6
ER -