Mitochondrial protein S-glutathionylation: an in-depth interrogation of the glutaredoxin-2 glutathionylome and its impact on bioenergetics and redox signaling

线粒体是哺乳动物细胞中ATP的主要来源，利用过氧化氢(H_2O_2)作为氧化还原信号的第二信使。过氧化氢的可获得性经过仔细的预算，以引起细胞对环境线索的快速反应，并避免氧化窘迫。在抗氧化剂防御和过氧化氢的抑制下，这些信号在产生激增后变得不敏感。蛋白质S谷胱甘肽基化是一种普遍存在的、可逆的、快速的氧化还原修饰，它涉及到抗氧化剂还原型谷胱甘肽(GSH)在蛋白质类半胱氨酸中的特定位置添加和移除。这些反应调节许多细胞功能，线粒体是控制燃料代谢、氧化磷酸化、溶质输入、分裂/融合和蛋白质折叠所必需的。我们在分离的雄性小鼠肝线粒体中发现，PGLU是丙酮酸脱氢酶(PDH)和α-酮戊二酸脱氢酶(KGDH)抑制H_2O_2产生的重要负反馈环。此外，我们发现它需要保护免受氧化损伤的诱导，并在GSH池的氧化还原缓冲能力恢复后被硫醇氧化还原酶谷氧还蛋白-2(GRX2)逆转。最后，我们还首次在线粒体PGLU中发现了一种新的性别二型性。雌性小鼠的肝脏和肌肉线粒体不需要GRX2-谷胱甘肽来抑制PDH和KGDH产生的过氧化氢，因为它具有优越的氧化还原缓冲能力和更有效的燃料代谢。我的研究项目的长期愿景是询问线粒体PGLU反应的功能，作为一个信号平台，将营养状态的变化和毒素的存在传达给细胞。然而，GRX2-谷胱甘肽从未被描述过，性别，一个营养信号和毒素清除的重要决定因素，对这一途径的影响也从未被研究过。这一建议旨在填补我们知识中的这些根本空白，并加深我们对GRX2-谷胱甘肽信号在线粒体代谢和氧化还原动态平衡中的作用的理解。这项提议的总体目标是利用一个结合转基因小鼠模型、原代细胞和培养细胞系的令人兴奋的平台来研究两条重要的研究路线。第一个目的是为了解决我最近提出的一个假设，即假设PGLU需要去激活线粒体H_2O_2信号。这将使用几个细胞株来研究GRX2，并评估过氧化氢介导的适应性信号通路的刺激。第二个目的是首次完整地描述GRX2-谷胱甘肽基因组的性别差异，以及2)GRX2缺失和性别对线粒体生物能量学和氧化还原缓冲能力的影响。这些发现将对我的长期研究愿景产生重大的积极影响。

Mitochondria are the main sources of ATP in mammalian cells and utilize hydrogen peroxide (H2O2) as a second messenger for redox signaling. H2O2 availability is budgeted carefully to elicit rapid cellular responses towards environmental cues and to avoid oxidative distress. These signals are desensitized following a burst in production by antioxidant defenses and through the inhibition of H2O2. Protein S-glutathionylation (PGLU) is a ubiquitous, reversible, and rapid redox modification that involves the site specific addition and removal of the antioxidant, reduced glutathione (GSH), to and from a proteinaceous cysteine. These reactions regulate many cellular functions and mitochondria are required to control fuel metabolism, oxidative phosphorylation, solute import, fission/fusion, and protein folding. We have discovered that PGLU serves as an important negative feedback loop for the inhibition of H2O2 production by pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase (KGDH) in liver mitochondria isolated from male mice. Additionally, we found that it is required to protect from the induction of oxidative distress and is reversed by the thiol oxidoreductase glutaredoxin-2 (GRX2) following restoration of the redox buffering capacity of the GSH pool. Finally, we also discovered a novel sexual dimorphism in mitochondrial PGLU for the first time. Liver and muscle mitochondria from female mice do no require the GRX2-glutathionylome to deactivate H2O2 production by PDH and KGDH due to a superior redox buffering capacity and more efficient fuel metabolism. The long-term vision for my research program is interrogate the function of mitochondrial PGLU reactions in serving as a signaling platform to communicate changes in nutritional status and the presence of toxins to cells. However, the GRX2-glutathionylome has never been characterized and the impact of sex, an important determinant in nutritional signaling and toxin elimination, on this pathway has never been examined. This proposal aims to fill these fundamental gaps in our knowledge and further our understanding of the role of GRX2-glutathionylome signaling in mitochondrial metabolism and redox homeostasis. The overall objective of this proposal is to investigate two important lines of inquiry using an exciting platform that combines transgenic mouse models, primary cells and cultured cell lines. The first aim is to address a recent hypothesis I generated that posited PGLU is required to deactivate mitochondrial H2O2 signals. This will be studied using several cell lines knocked down for GRX2 and assessing the H2O2-mediated stimulation of adaptative signaling pathways. The second aim is to fully 1) characterize sex differences in the GRX2-glutathionylome for the first time and 2) interrogate the effect of both the loss of GRX2 and sex on mitochondrial bioenergetics and redox buffering capacity. These findings will have a significant positive impact for my long-term research vision.