Complexities of the chemogenetic toolkit: differential mDAAO activation by D-amino substrates and subcellular targeting

Erdoğan, Yusuf Ceyhun and Altun, Hamza Yusuf and Seçilmiş, Melike and Ata, Büşra Nur and Sevimli, Gülşah and Çokluk, Zeynep and Ghaffari Zaki, Asal and Sezen, Serap and Akgul Caglar, Tuba and Sevgen, İlker and Steinhorn, Benjamin and Ai, Huiwang and Öztürk, Gürkan and Belousov, Vsevelod V. and Michel, Thomas and Eroğlu, Emrah (2021) Complexities of the chemogenetic toolkit: differential mDAAO activation by D-amino substrates and subcellular targeting. Free Radical Biology and Medicine, 177 . pp. 132-142. ISSN 0891-5849 (Print) 1873-4596 (Online)

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A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H2O2 to living cells or tissues. However, the addition of H2O2 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 H2O2 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 H2O2 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 H2O2 under hypoxic conditions imaged with the novel ultrasensitive H2O2 sensor (HyPer7). Moreover, this approach was suitable for generating H2O2 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 H2O2 generation. When paired with the hydrogen sulfide (H2S) sensor hsGFP, administration of the sulfur-containing amino acid D-cysteine to cells expressing mDAAO generates robust H2S signals. We also show that chemogenetic H2O2 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.
Item Type: Article
Uncontrolled Keywords: D-amino Acid oxidase (DAAO) catalysis and targeting; D-amino acids; Genetically encoded biosensors; hsGFP; Hydrogen peroxide; Hydrogen sulfide; HyPer7; Multiparametric imaging
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Sabancı University Nanotechnology Research and Application Center
Depositing User: Emrah Eroğlu
Date Deposited: 28 Aug 2022 10:06
Last Modified: 28 Aug 2022 10:06

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