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，并利用过氧化氢（H2 O2）作为氧化还原信号的第二信使。H2 O2的可用性是仔细预算，以引起对环境线索的快速细胞反应，并避免氧化应激。这些信号在抗氧化剂防御和H2 O2抑制的生产爆发后脱敏。蛋白S-谷胱甘肽化（PGLU）是一种普遍存在的、可逆的、快速的氧化还原修饰，其涉及抗氧化剂还原型谷胱甘肽（GSH）与蛋白半胱氨酸的位点特异性添加和去除。这些反应调节许多细胞功能，并且需要线粒体来控制燃料代谢、氧化磷酸化、溶质输入、裂变/融合和蛋白质折叠。我们已经发现PGLU作为一个重要的负反馈回路抑制H2 O2的生产丙酮酸脱氢酶（PDH）和α-酮戊二酸脱氢酶（KGDH）在肝线粒体分离的雄性小鼠。此外，我们发现，它需要保护免受氧化应激的诱导，并且在还原型谷胱甘肽池的氧化还原缓冲能力恢复后，通过巯基氧化还原酶谷氧还蛋白-2（GRX 2）逆转。最后，我们还首次在线粒体PGLU中发现了一种新的两性异形现象。由于上级氧化还原缓冲能力和更有效的燃料代谢，来自雌性小鼠的肝脏和肌肉线粒体不需要GRX 2-谷胱甘肽组来使PDH和KGDH产生的H2 O2失活。我的研究计划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，以传达营养状态的变化和细胞毒素的存在。然而，GRX 2-谷胱甘肽组从来没有被表征，性别的影响，营养信号和毒素消除的一个重要决定因素，对这一途径从未被检查。该提案旨在填补我们知识中的这些基本空白，并进一步了解GRX 2-谷胱甘肽信号在线粒体代谢和氧化还原稳态中的作用。该提案的总体目标是使用一个令人兴奋的平台，结合转基因小鼠模型，原代细胞和培养细胞系，研究两个重要的研究方向。第一个目的是解决我最近提出的一个假设，即假定PGLU是灭活线粒体H2 O2信号所必需的。这将使用几种GRX 2敲低的细胞系进行研究，并评估H2 O2介导的适应性信号传导途径的刺激。第二个目的是充分1）首次表征GRX 2-谷胱甘肽组的性别差异，2）询问GRX 2和性别的丧失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.