School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore.
Redox Biol. 2019 Jun;24:101168. doi: 10.1016/j.redox.2019.101168. Epub 2019 Mar 14.
Reactive oxygen species (ROS) induce different cellular stress responses but can also mediate cellular signaling. Augmented levels of ROS are associated with aging, cancer as well as various metabolic and neurological disorders. ROS can also affect the efficacy and adverse effects of drugs. Although proteins are key mediators of most ROS effects, direct studies of ROS-modulated-protein function in the cellular context are very challenging. Therefore the understanding of specific roles of different proteins in cellular ROS responses is still relatively rudimentary. In the present work we show that Mass Spectrometry-Cellular Thermal Shift Assay (MS-CETSA) can directly monitor ROS and redox modulations of protein structure at the proteome level. By altering ROS levels in cultured human hepatocellular carcinoma cell lysates and intact cells, we detected CETSA responses in many proteins known to be redox sensitive, and also revealed novel candidate ROS sensitive proteins. Studies in intact cells treated with hydrogen peroxide and sulfasalazine, a ROS modulating drug, identified not only proteins that are directly modified, but also proteins reporting on downstream cellular effects. Comprehensive changes are seen on rate-limiting proteins regulating key cellular processes, including known redox control systems, protein degradation, epigenetic control and protein translational processes. Interestingly, concerted shifts on ATP-binding proteins revealed redox-induced modulation of ATP levels, which likely control many cellular processes. Collectively, these studies establish CETSA as a novel method for cellular studies of redox modulations of proteins, which implicated in a wide range of processes and for the discovery of CETSA-based biomarkers reporting on the efficacy as well as adverse effects of drugs.
活性氧（ROS）诱导不同的细胞应激反应，但也可以介导细胞信号转导。ROS 水平的增加与衰老、癌症以及各种代谢和神经紊乱有关。ROS 还会影响药物的疗效和不良反应。尽管蛋白质是大多数 ROS 效应的关键介质，但在细胞环境中直接研究 ROS 调节的蛋白质功能具有很大的挑战性。因此，对不同蛋白质在细胞 ROS 反应中的特定作用的理解仍然相对原始。在本工作中，我们表明，质谱细胞热转移分析（MS-CETSA）可以直接监测蛋白质组水平的 ROS 和氧化还原修饰对蛋白质结构的影响。通过改变培养的人肝癌细胞裂解物和完整细胞中的 ROS 水平，我们在许多已知对氧化还原敏感的蛋白质中检测到 CETSA 反应，并且还揭示了新的候选 ROS 敏感蛋白质。用过氧化氢和柳氮磺胺吡啶（一种 ROS 调节药物）处理完整细胞的研究不仅鉴定了直接修饰的蛋白质，还鉴定了报告下游细胞效应的蛋白质。在调节关键细胞过程的限速蛋白上观察到全面的变化，包括已知的氧化还原控制系统、蛋白质降解、表观遗传控制和蛋白质翻译过程。有趣的是，ATP 结合蛋白的协同变化揭示了氧化还原诱导的 ATP 水平调节，这可能控制着许多细胞过程。总的来说，这些研究确立了 CETSA 作为一种研究蛋白质氧化还原修饰的新方法，该方法涉及广泛的过程，并发现了基于 CETSA 的生物标志物，可报告药物的疗效和不良反应。
