The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.神经元分化的初始阶段对神经元功能的正常发挥至关重要。用于研究该初始阶段的极具参考价值的模型为视黄酸刺激的SH-SY5Y细胞。尽管此类分化进程与氧化还原敏感过程密切相关，但目前学界尚未明确细胞蛋白质组如何调控氧化还原动态变化以及活性氧（reactive oxygen species, ROS）的代谢管理。本研究通过定量氧化还原蛋白质组学（quantitative redox proteomics）技术，绘制了SH-SY5Y细胞的全局半胱氨酸氧化还原修饰全景图谱。研究结果显示，氧化还原修饰改变在分化早期即已发生，并会影响神经元标志物蛋白的表达与神经突的延伸。对活性氧的时空特征分析表明，视黄酸刺激后2小时，细胞质中超氧阴离子/过氧化氢的生成会出现依赖于NOX2（NADPH氧化酶2）的峰值。在该时间节点，275个半胱氨酸氧化还原状态发生改变的蛋白质中，有241个可在视黄酸刺激下发生可逆氧化修饰。本研究还精准定位了参与视黄酸稳态调控与细胞骨架动力学的蛋白质的氧化还原修饰改变情况。