Superoxide-responsive quinone methide precursors (QMP-SOs) to study superoxide biology by proximity labeling and chemoproteomics†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-08-07 DOI:10.1039/D4CB00111G
Hinyuk Lai and Clive Yik-Sham Chung
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Abstract

Superoxide is a reactive oxygen species (ROS) with complex roles in biological systems. It can contribute to the development of serious diseases, from aging to cancers and neurodegenerative disorders. However, it can also serve as a signaling molecule for important life processes. Monitoring superoxide levels and identifying proteins regulated by superoxide are crucial to enhancing our understanding of this growing field of redox biology and signaling. Given the high reactivity and very short lifetime of superoxide compared to other ROS in biological systems, proteins redox-modified by superoxide should be in close proximity to where superoxide is generated endogenously, i.e. superoxide hotspots. This inspires us to develop superoxide-specific quinone methide-based precursors, QMP-SOs, for proximity labeling of proteins within/near superoxide hotspots to image superoxide and profile proteins associated with superoxide biology by chemoproteomics. QMP-SOs specifically react with superoxide to generate an electrophilic quinone methide intermediate, which subsequently reacts with nucleophilic amino acids to induce a covalent tag on proteins, as revealed by liquid chromatography-mass spectrometry (LC-MS) and shotgun MS experiments. The alkyne handle on the covalent tag enables installation of fluorophores onto the tagged proteins for fluorescence imaging of superoxide in cells under oxidative stress. By establishing a chemoproteomics platform, QMP-SO-TMT, we identify DJ-1 and DLDH as proteins associated with superoxide biology in liver cancer cells treated with menadione. This work should provide insights into the crosstalk between essential cellular events and superoxide redox biology, as well as the design principles of quinone methide-based probes to study redox biology through proximity labeling and chemoproteomics.

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通过近距离标记和化学蛋白质组学研究超氧化物生物学的超氧化物反应性醌甲醚前体(QMP-SOs)
超氧化物是一种活性氧(ROS),在生物系统中发挥着复杂的作用。它可导致严重疾病的发生,包括衰老、癌症和神经退行性疾病。不过,它也可以作为重要生命过程的信号分子。监测超氧化物水平并确定受超氧化物调控的蛋白质,对于加深我们对这一不断发展的氧化还原生物学和信号转导领域的了解至关重要。与生物系统中的其他 ROS 相比,超氧化物具有高反应性和极短的生命周期,因此被超氧化物氧化还原修饰的蛋白质应该靠近超氧化物内源生成的地方,即超氧化物热点。这启发我们开发了超氧化物特异性甲脒醌前体 QMP-SOs,用于近距离标记超氧化物热点内/附近的蛋白质,通过化学蛋白质组学对超氧化物进行成像,并分析与超氧化物生物学相关的蛋白质。QMP-SOs 能与超氧化物发生特异性反应,生成亲电的甲脒醌中间体,随后与亲核氨基酸发生反应,诱导蛋白质上的共价标签,液相色谱-质谱(LC-MS)和霰粒质谱(shotgun MS)实验显示了这一点。共价标签上的炔柄可以在标签蛋白质上安装荧光团,对氧化应激细胞中的超氧化物进行荧光成像。通过建立化学蛋白质组学平台 QMP-SO-TMT,我们鉴定出 DJ-1 和 DLDH 是与经甲萘醌处理的肝癌细胞中超氧化物生物学相关的蛋白质。这项工作将有助于深入了解细胞基本事件与超氧化物氧化还原生物学之间的相互关系,并为通过近距离标记和化学蛋白质组学研究氧化还原生物学提供基于甲喹酮探针的设计原则。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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