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.