Dissecting in vivo and in vitro redox responses using chemogenetics.

Hydrogen peroxide (H2O2) is the most abundant reactive oxygen species (ROS) within mammalian cells. At low concentrations, H2O2 serves as a versatile cell signaling molecule that mediates vital physiological functions. Yet at higher concentrations, H2O2 can be a toxic molecule by promoting pathological oxidative stress in cells and tissues. Within normal cells, H2O2 is differentially distributed in a variety of subcellular locales. Moreover, many redox-active enzymes and their substrates are themselves differentially distributed within cells. Numerous reports have described the biological and biochemical consequences of adding exogenous H2O2 to cultured cells and tissues, but many of these observations are difficult to interpret: the effects of exogenous H2O2 do not necessarily replicate the cellular responses to endogenous H2O2. In recent years, chemogenetic approaches have been developed to dynamically regulate the abundance of H2O2 in specific subcellular locales. Chemogenetic approaches have been applied in multiple experimental systems, ranging from in vitro studies on the intracellular transport and metabolism of H2O2, all the way to in vivo studies that generate oxidative stress in specific organs in living animals. These chemogenetic approaches have exploited a yeast-derived d-amino acid oxidase (DAAO) that synthesizes H2O2 only in the presence of its d-amino acid substrate. DAAO can be targeted to various subcellular locales, and can be dynamically activated by the addition or withdrawal of its d-amino acid substrate. In addition, recent advances in the development of highly sensitive genetically encoded H2O2 biosensors are providing a better understanding of both physiological and pathological oxidative pathways. This review highlights several applications of DAAO as a chemogenetic tool across a wide range of biological systems, from analyses of subcellular H2O2 metabolism in cells to the development of new disease models caused by oxidative stress in vivo.