Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.
Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.
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DOI：
10.1016/j.freeradbiomed.2021.10.023
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发表时间：
2021-12
影响因子：
7.4
通讯作者：
Eroglu E
中科院分区：
文献类型：
--
作者：
Erdogan YC;Altun HY;Secilmis M;Ata BN;Sevimli G;Cokluk Z;Zaki AG;Sezen S;Akgul Caglar T;Sevgen İ;Steinhorn B;Ai H;Öztürk G;Belousov VV;Michel T;Eroglu E
研究氧化剂调节的细胞内途径的常见方法是向活细胞或组织中添加外源性 H2O2。然而，向细胞或组织的培养基中添加 H2O2 的方法并不能准确复制细胞内氧化还原介导的细胞反应。基于 D-氨基酸氧化酶 (DAAO) 的化学遗传学工具代表了信息丰富的方法学进展，通过提供或提取 DAAO 底物 D-氨基酸，可以以高时空分辨率按需生成 H2O2。通过使用 DAAO 进行的研究，人们对 H2O2 的细胞内转运有了很多了解，但这些有价值的工具在许多培养细胞中仍未完全表征。在这项研究中，我们详细描述和表征了具有改进的催化活性的 DAAO 的新修饰变体（称为 mDAAO）的特征。我们使用活细胞成像技术在几种培养细胞系中测试了 mDAAO 功能。我们的成像实验表明，mDAAO 适合在缺氧条件下生成 H2O2，并使用新型超灵敏 H2O2 传感器 (HyPer7) 进行成像。此外，这种方法适合在亚细胞区域以可逆且浓度依赖性的方式产生 H2O2。此外，我们发现 D-氨基酸的选择对 mDAAO 依赖性细胞内 H2O2 产生有不同的影响。当与硫化氢 (H2S) 传感器 hsGFP 配对时，向表达 mDAAO 的细胞施用含硫氨基酸 D-半胱氨酸会产生强大的 H2S 信号。我们还表明，不同细胞类型中化学遗传学 H2O2 的产生会产生不同的 HyPer7 谱。这些研究充分描述了新的 mDAAO 作为一种新型化学遗传学工具的特点，并为细胞操作提供了多参数方法，这可能为氧化还原生物化学家开辟新的研究路线，以高空间和时间精度剖析 ROS 信号通路的作用。
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.
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影响因子：
16.6
作者：
Eroglu E;Gottschalk B;Charoensin S;Blass S;Bischof H;Rost R;Madreiter-Sokolowski CT;Pelzmann B;Bernhart E;Sattler W;Hallström S;Malinski T;Waldeck-Weiermair M;Graier WF;Malli R
通讯作者：
Malli R
影响因子：
48
作者：
Cameron, William D.;Bui, Cindy V.;Rocheleau, Jonathan V.
通讯作者：
Rocheleau, Jonathan V.
影响因子：
48
作者：
Gutscher, Marcus;Pauleau, Anne-Laure;Dick, Tobias P.
通讯作者：
Dick, Tobias P.
影响因子：
16.6
作者：
Ermakova YG;Bilan DS;Matlashov ME;Mishina NM;Markvicheva KN;Subach OM;Subach FV;Bogeski I;Hoth M;Enikolopov G;Belousov VV
通讯作者：
Belousov VV
影响因子：
3.4
作者：
Fernández-Lafuente, R;Rodriguez, V;Guisán, JM
通讯作者：
Guisán, JM