Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting

Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

“…As shown in Figure 2B, administration of 10 mM D-alanine to endothelial cells co-expressing targeted mCherry-mDAAO and HyPer7 triggered a detectable yet sluggish H2O2 signal in the cell cytosol. Our group's previous study showed that 10 mM D-alanine triggered robust and fast H2O2 generation in different cell types (i.e., HEK293T or U87MG) [24]. This observation suggests that endothelial cells hardly take up D-alanine, probably due to a tightly regulated membrane trafficking and import/export mechanisms in these cell types.…”

“…Endothelial cells are hardly transfectable cells with conventional transfection reagents [30]. Therefore, this study generated EA.hy926 cell lines stably expressing yeast-derived (from R. gracilis) D-amino acid oxidasetermed mDAAO -fused to a red fluorescent protein (mCherry) [24] using lentivirus approaches as described previously [27]. Purified lentiviral particles encoding for cytosolic targeted mCherry-mDAAO chimera were used to infect endothelial cells.…”

“…In recent years, the toolkit of redox biologists has been expanded by a yeast-derived enzyme D-amino acid oxidase (DAAO), which turned useful as a chemogenetic tool permitting the manipulation of redox levels on the level of tissues, cells, and even subcellular locales with high precision [22][23][24]. Chemogenetics refers to an experimental system that becomes activated in the presence of its substrate and remains silent in the absence of its biochemical stimulus [25].…”

“…One mole of D-amino acid produces equimolar concentrations of H2O2 [26]. The type of D-amino acid and the amino acid uptake efficiency dictates the intracellular generation of mDAAO's byproducts (Figure 1A), which typically yields H2O2 in the lower micromolar range [24].…”

Mitochondrial H2O2release does not directly cause genomic DNA damage

“…Total DAAO activity in these cell lines is likely determined by a combination of the level of DAAO expression, folding, accessibility, cofactor (FAD) availability as well as substrate (D-amino acid) levels, uptake, and diffusion 20 . Because DAAO uses equimolar amounts of molecular oxygen to produce H 2 O 2 , we assessed DAAO activity by measuring the oxygen consumption rate (OCR) in response to titration with [D-Ala].…”