A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous HO to living cells or tissues. However, the addition of HO to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of HO on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids. Much has been learned about the intracellular transport of HO through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of HO under hypoxic conditions imaged with the novel ultrasensitive HO sensor (HyPer7). Moreover, this approach was suitable for generating HO in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of d-amino acids differentially affects mDAAO-dependent intracellular HO generation. When paired with the hydrogen sulfide (HS) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust HS signals. We also show that chemogenetic HO generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision.
Copyright © 2021. Published by Elsevier Inc.