AIMS: Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2. RESULTS: We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci. INNOVATION: Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells. CONCLUSION: Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.
AIMS: Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2. RESULTS: We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci. INNOVATION: Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells. CONCLUSION: Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.
Authors: Ágnes Donkó; Stanislas Morand; Agnieszka Korzeniowska; Howard E Boudreau; Melinda Zana; László Hunyady; Miklós Geiszt; Thomas L Leto Journal: Free Radic Biol Med Date: 2014-05-20 Impact factor: 7.376