How much H2O2 is produced by recombinant D-Amino acid oxidase in mammalian cells?

Mikhail E. Matlashov, Vsevolod V. Belousov, Grigori Enikolopov

Research output: Contribution to journalReview articlepeer-review

35 Scopus citations

Abstract

Yeast D-amino acid oxidase (DAO) can serve as a genetically encoded producer of reactive oxygen species (ROS) in redox signaling studies. However, dynamics of hydrogen peroxide production and its sensitivity to externally added D-alanine (D-Ala) in cells have not been determined. Here we show that DAO, fused to a genetically encoded H2O2 indicator HyPer, can be used for controlled production of ROS in living eukaryotic cells. We found a clear heterogeneity in ROS production dynamics between individual cells. Moreover, different cell lines demonstrated distinct sensitivity to added D-Ala. Finally, by comparing signals generated by the HyPer-DAO fusion protein versus coexpressed HyPer and DAO proteins, we show that the fusion system is more sensitive to hydrogen peroxide production. Our results show the utility of the HyPer-DAO genetically encoded system for redox signaling studies and suggest that H2O2 produced by DAO in the cytoplasm acts locally in close proximity to the enzyme. Antioxid. Redox Signal. 20, 1039-1044.

Original languageEnglish (US)
Pages (from-to)1039-1044
Number of pages6
JournalAntioxidants and Redox Signaling
Volume20
Issue number7
DOIs
StatePublished - Mar 1 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology

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