Figure 4.

Comparison of conventional versus chemogenetic approaches to perturb cellular redox balance. (Left) Conventional approach to perturb intracellular redox balance by adding exogenous hydrogen peroxide (H₂O₂) or redox-active small molecules to cells or tissues. The direct addition of H₂O₂ to cells has highly variable effects on intracellular pathways due to the variable permeability of cell membranes to H₂O₂ and the differential distribution of H₂O₂ between intracellular organelles. Moreover, the concentrations of exogenous H₂O₂ needed to activate physiological pathways can also produce toxic effects on cells. Redox-active small molecules can also be added to cells, with variable (and often nonspecific) effects on intracellular oxidants. (Right) A chemogenetic approach that permits dynamic regulation of H₂O₂ generated by recombinant yeast d-amino acid oxidase (DAAO) expressed in mammalian cells. H₂O₂ can be reversibly generated in specific intracellular organelles by providing (or withholding) d-amino acids. DAAO can be cloned as a fusion protein along with the H₂O₂ biosensor HyPer to document H₂O₂ formation within specific subcellular compartments. Alternatively, the DAAO and HyPer can be differentially targeted to distinct subcellular organelles to study intracellular H₂O₂ diffusion from the site of H₂O₂ synthesis by DAAO to the organelle in which H₂O₂ is detected by HyPer. Chemogenetic approaches have been used both in cultured cells and in animals in vivo to dissect both physiological and pathophysiological oxidant-modulated pathways. Chemogenetic methods have enabled experimenters to isolate redox state as an independent variable of both in vitro and in vivo systems. Moreover, biosensors can be targeted to compartments distinct from the organelle expressing DAAO, enabling the experimenter to study the effects of oxidants generated in different subcellular locales. Figure adapted from images created with BioRender.com.