Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.
Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.
复制标题
用于实时 H2O2 监测的快速基因编码传感器的结构引导工程。
DOI：
10.1101/2024.01.31.578117
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Chavki
中科院分区：
文献类型：
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作者：
Lee,JustinDaho;Won,Woojin;Kimball,Kandace;Wang,Yihan;Yeboah,Fred;Evitts,KiraM;Neiswanger,Carlie;Schattauer,Selena;Rappleye,Michael;Bremner,SamanthaB;Chun,Changho;Smith,Netta;Mack,DavidL;Young,JessicaE;Lee,CJustin;Chavki
过氧化氢(H_2O_2)在生理和病理中具有多效性，是氧化还原生物学中的中心氧化剂。然而，实时监测活细胞和组织中的过氧化氢仍然是一个挑战。我们利用细菌过氧化氢结合结构域OxyR，开发了一种光遗传过氧化氢传感器(OROS)来解决这一缺口。以前设计的基于OxyR的荧光过氧化氢传感器缺乏有效实时监测所需的灵敏度或响应速度。通过对大肠杆菌ecOxyR与环状置换的绿色荧光蛋白(CpGFP)的融合进行结构重新设计，我们创造了一种新型的绿色荧光过氧化氢传感器Oros-G。OROS-G具有高灵敏度和快速的开关动力学，是监测细胞内过氧化氢动态的理想选择。我们成功地跟踪了不同生物系统中的实时瞬时和稳态过氧化氢水平，包括人干细胞来源的神经元和心肌细胞，原代神经元和星形胶质细胞，以及小鼠神经元和星形胶质细胞在体外脑片中。这些应用表明，Oros有能力监测过氧化氢作为对药物诱导的氧化应激、G蛋白偶联受体(GPCR)诱导的细胞信号的二级反应，以及在适应不同的代谢应激时的能力。我们通过β-putrisine-MAOB轴显示了星形胶质细胞中氧化应激的增加，强调了该传感器在验证神经退行性疾病模型中的相关性。OROS是一个多用途的工具，提供了一个了解过氧化氢信号动态图景的窗口。这一进展为更深入地理解氧化还原生理学铺平了道路，对与氧化应激相关的疾病，如癌症、神经退行性疾病和心血管疾病具有重要意义。
Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor’s relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.