How Much H₂O₂ 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 H 2 O 2 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 H 2 O 2 produced by DAO in the cytoplasm acts locally in close proximity to the enzyme.

Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress

“…While it is uncontested that catalase is located in the peroxisomal matrix, other locations of catalase activity are also relevant, such as the mitochondria [104] and even the extracellular space [105]. Using D-amino acid oxidase as genetically encoded producer of H 2 O 2 , in conjunction with the fused HyPer probe, it becomes possible to examine intracellular H 2 O 2 dynamics quantitatively [106]·H 2 O 2 microdomains in receptor tyrosine kinase signaling are now amenable to imaging [107].…”

Using Sensors and Generators of H2O2to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

“…This involves manipulating the antioxidant level through reaction with small molecules and genetic overexpression of protein generators that can produce specific oxidants at controlled rates intracellularly. D-amino acid oxidase (DAAO) from Rhodotorula gracilis has been previously shown to be a powerful intracellular enzyme that can produce tunable and localizable H 2 O 2 intracellularly depending on the concentration of D-alanine (D-Ala) substrate added (18,20,33). Using DAAO to provide spatial and temporal control over the production of H 2 O 2 , we can mimic the generation of the oxidant by chemotherapeutics and isolate its contribution to the mechanism of toxicity, if any.…”

Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor

“…Thus, microscopic analysis is the preferred measurement technique because it leaves the samples unperturbed. Most prior studies that included microscopic image analysis of cells expressing peroxide sensors used small sample sizes (~10 cells) (19,20,(29)(30)(31)(32). To facilitate analysis of larger sample sizes, we automated the image analysis process, using a combination of ImageJ (National Institutes of Health, Bethesda, MD) and CellProfiler software (Broad Institute).…”