title: How much H(2)O(2) is produced by recombinant D-amino acid oxidase in mammalian cells?
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.
authors.affiliation: 1 NBIC, Moscow Institute of Physics and Technology , Moscow, Russia .
authors.forename: Mikhail E
authors.initials: ME
authors.lastname: Matlashov
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Belousov
authors.forename: Grigori
authors.initials: G
authors.lastname: Enikolopov
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase
references.citation: Int J Cancer. 2008 Mar 1;122(5):1135-44
references.pmid: 17990314
references.citation: Nature. 2009 Jun 18;459(7249):996-9
references.pmid: 19494811
references.citation: Cell Mol Life Sci. 2007 Jun;64(11):1373-94
references.pmid: 17396222
references.citation: Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17385-90
references.pmid: 20855618
references.citation: Nat Chem Biol. 2008 May;4(5):278-86
references.pmid: 18421291
references.citation: Nat Methods. 2006 Apr;3(4):281-6
references.pmid: 16554833
references.citation: J Mol Biol. 2002 Nov 29;324(3):535-46
references.pmid: 12445787
references.citation: Mol Cell. 2012 Sep 14;47(5):767-76
references.pmid: 22819323