Spatial and temporal analysis of NADPH oxidase-generated hydrogen peroxide signals by novel fluorescent reporter proteins

Antioxid Redox Signal. 2013 Aug 20;19(6):523-34. doi: 10.1089/ars.2012.4594. Epub 2012 Dec 26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques
  • COS Cells
  • Chlorocebus aethiops
  • Dual Oxidases
  • Fluorescence Resonance Energy Transfer
  • Glutathione Peroxidase / biosynthesis
  • Glutathione Peroxidase / genetics
  • Green Fluorescent Proteins / biosynthesis*
  • Green Fluorescent Proteins / genetics
  • HeLa Cells
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Knockout
  • NADPH Oxidase 2
  • NADPH Oxidases / metabolism*
  • Oxidation-Reduction
  • Paracrine Communication
  • Protein Transport
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae Proteins / biosynthesis
  • Saccharomyces cerevisiae Proteins / genetics
  • Signal Transduction
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics


  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • YAP1 protein, S cerevisiae
  • Green Fluorescent Proteins
  • Hydrogen Peroxide
  • Dual Oxidases
  • Glutathione Peroxidase
  • HYR1 protein, S cerevisiae
  • CYBB protein, human
  • NADPH Oxidase 2
  • NADPH Oxidases
  • DUOX1 protein, human