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

Autor: Erdogan YC; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Altun HY; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Secilmis M; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Ata BN; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Sevimli G; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Cokluk Z; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Zaki AG; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Sezen S; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Akgul Caglar T; Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey., Sevgen İ; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey., Steinhorn B; Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA., Ai H; Department of Molecular Physiology and Biological Physics, Center for Membrane and Cell Physiology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA., Öztürk G; Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey; Physiology Department, International School of Medicine, Istanbul Medipol University, Istanbul, Turkey., Belousov VV; Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, 117997, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, Moscow, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, 117997, Moscow, Russia; Institute of Cardiovascular Physiology, Universitätsmedizin Göttingen, 37073, Göttingen, Germany., Michel T; Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. Electronic address: thomas_michel@hms.harvard.edu., Eroglu E; Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey; Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Austria; Nanotechnology Research and Application Center, Sabanci University, Istanbul, Turkey. Electronic address: emrah.eroglu@sabanciuniv.edu.
Jazyk: angličtina
Zdroj: Free radical biology & medicine [Free Radic Biol Med] 2021 Dec; Vol. 177, pp. 132-142. Date of Electronic Publication: 2021 Oct 20.
DOI: 10.1016/j.freeradbiomed.2021.10.023
Abstrakt: A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H 2 O 2 to living cells or tissues. However, the addition of H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 under hypoxic conditions imaged with the novel ultrasensitive H 2 O 2 sensor (HyPer7). Moreover, this approach was suitable for generating H 2 O 2 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 H 2 O 2 generation. When paired with the hydrogen sulfide (H 2 S) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust H 2 S signals. We also show that chemogenetic H 2 O 2 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 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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