Molecular Cell Manuscript_Zhiwei et al., 2017

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The Mitochondrial Calcium Uniporter (MCU) complex is the major pathway by which these signals are integrated in the mitochondria. Whether and how these coactive elements interact with MCU has not been established. As an approach towards understanding the regulation of MCU channel activity by oxidative milieu, we adapted inflammatory and hypoxia model systems. Our studies identified the conserved cysteine at position 97 to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher-order oligomer formation in the inner mitochondrial membrane. Both oxidation and mutation of MCU Cys-97 exhibited persistent activation of the MCU channel with higher [Ca2+]m uptake rate, increased IMCU, elevated mitochondrial ROS and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulatory components. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

钙离子动态变化与氧化信号通路是线粒体生物能学与细胞功能的核心机制。线粒体钙单向转运体（Mitochondrial Calcium Uniporter, MCU）复合物是上述信号在线粒体中整合的主要途径。目前尚未明确这些协同作用因子是否以及如何与MCU发生相互作用。为探究氧化微环境对MCU通道活性的调控机制，我们采用了炎症与缺氧模型系统开展研究。本研究鉴定出人类MCU中第97位保守半胱氨酸是唯一可发生S-谷胱甘肽化修饰的反应性巯基。进一步的生化、结构与超分辨率成像分析显示，MCU的氧化修饰可促进其在线粒体内膜上形成高级寡聚体。无论是MCU的氧化修饰还是Cys-97位点突变，均能使MCU通道持续激活，表现为更高的线粒体基质钙离子摄取速率、增强的MCU电流、升高的线粒体活性氧水平，以及加剧的线粒体基质钙离子超载诱导的细胞死亡。相反，上述效应基本不依赖于MCU与其调控组分的相互作用。本研究揭示了Cys-97位点在活性氧感知与MCU活性调控中的独特功能角色。