Abstract
Hydrogen peroxide (H(2)O(2)) is the most abundant reactive oxygen species (ROS) within mammalian cells. At low concentrations, H(2)O(2) serves as a versatile cell signaling molecule that mediates vital physiological functions. Yet at higher concentrations, H(2)O(2) can be a toxic molecule by promoting pathological oxidative stress in cells and tissues. Within normal cells, H(2)O(2) 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 H(2)O(2) to cultured cells and tissues, but many of these observations are difficult to interpret: the effects of exogenous H(2)O(2) do not necessarily replicate the cellular responses to endogenous H(2)O(2). In recent years, chemogenetic approaches have been developed to dynamically regulate the abundance of H(2)O(2) 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 H(2)O(2), 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) metabolism in cells to the development of new disease models caused by oxidative stress in vivo.