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

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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.