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

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dc.authorid0000-0003-2033-860X
dc.authorid0000-0003-0352-1947
dc.contributor.authorErdoğan, Yusuf Can
dc.contributor.authorAltun, Hamza Yusuf
dc.contributor.authorSeçilmiş, Melike
dc.contributor.authorAta, Büşra N.
dc.contributor.authorSevimli, Gülşah
dc.contributor.authorÇokluk, Zeynep
dc.contributor.authorZaki, Asal Ghaffari
dc.contributor.authorSezen, Serap
dc.contributor.authorAkgül Çağlar, Tuba
dc.contributor.authorSevgen, İlker
dc.contributor.authorSteinhorn, Benjamin
dc.contributor.authorAi, Huiwang
dc.contributor.authorÖztürk, Gürkan
dc.contributor.authorBelousov, Vsevelod V.
dc.contributor.authorMichel, Thomas
dc.contributor.authorEroğlu, Emrah
dc.date.accessioned2021-11-05T06:23:01Z
dc.date.available2021-11-05T06:23:01Z
dc.date.issued2021
dc.departmentİstanbul Medipol Üniversitesi, Rektörlük, Sağlık Bilim ve Teknolojileri Araştırma Enstitüsü
dc.departmentİstanbul Medipol Üniversitesi, Uluslararası Tıp Fakültesi, Temel Tıp Bilimleri Bölümü, Fizyoloji Ana Bilim Dalı
dc.description.abstractA 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.
dc.identifier.citationErdoğan, Y. C., Altun, H. Y., Seçilmiş, M., Ata, B. N., Sevimli, G., Çokluk, Z. ... Eroğlu, E. (2021). Complexities of the chemogenetic toolkit: Differential mDAAO activation by D-amino substrates and subcellular targeting. Free Radical Biology and Medicine, 177, 132-142. https://dx.doi.org/10.1016/j.freeradbiomed.2021.10.023
dc.identifier.doi10.1016/j.freeradbiomed.2021.10.023
dc.identifier.endpage142
dc.identifier.issn0891-5849
dc.identifier.issn1873-4596
dc.identifier.scopusqualityQ1
dc.identifier.startpage132
dc.identifier.urihttps://dx.doi.org/10.1016/j.freeradbiomed.2021.10.023
dc.identifier.urihttps://hdl.handle.net/20.500.12511/8565
dc.identifier.volume177
dc.identifier.wosqualityQ1
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakScopus
dc.indekslendigikaynakPubMed
dc.language.isoen
dc.publisherElsevier Inc.
dc.relation.ispartofFree Radical Biology and Medicineen_US
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanı
dc.rightsinfo:eu-repo/semantics/embargoedAccess
dc.subjectD-Amino Acid Oxidase (DAAO) Catalysis and Targeting
dc.subjectD-Amino Acids
dc.subjectGenetically Encoded Biosensors
dc.subjecthsGFP
dc.subjectHydrogen Peroxide
dc.subjectHydrogen Sulfide
dc.subjectHyPer7
dc.subjectMultiparametric Imaging
dc.titleComplexities of the chemogenetic toolkit: Differential mDAAO activation by D-amino substrates and subcellular targeting
dc.typeArticle

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