Yeast Sir2 is an NAD-dependent histone deacetylase related to oxidative stress and aging. In a previous study, we showed that Sir2 is regulated by S-glutathionylation of key cysteine residues located at the catalytic domain. Mutation of these residues results in strains with increased resistance to disulfide stress. In the present study, these mutant cells were highly resistant to acetic acid and had an increased chronological life span. Mutant cells had increased acetyl-CoA synthetase activity, which converts acetic acid generated by yeast metabolism to acetyl.CoA. This could explain the acetic acid resistance and lower levels of this toxic acid in the extracellular media during aging. Increased acetyl-CoA levels would raise lipid droplets, a source of energy during aging, and fuel glyoxylate-dependent gluconeogenesis. The key enzyme of this pathway, phosphoenolpyruvate carboxykinase (Pck1), showed increased activity in these Sir2 mutant cells during aging. Sir2 activity decreased when cells shifted to the diauxic phase in the mutant strains, compared to the WT strain. Since Pck1 is inactivated through Sir2-dependent deacetylation, the decline in Sir2 activity explained the rise in Pck1 activity. As a consequence, storage of sugars such as trehalose would increase. We conclude that extended longevity observed in the mutants was a combination of increased lipid droplets and trehalose, and decreased acetic acid in the extracellular media. These results offer a deeper understanding of the redox regulation of Sir2 in acetic acid resistance, which is relevant in some food and industrial biotechnology and also in the metabolism associated to calorie restriction, aging and pathologies such as diabetes.

The regulatory mechanisms of yeast Sir2, the founding member of the sirtuin family involved in oxidative stress and aging, are unknown. Redox signaling controls many cellular functions, especially under stress situations, with dithiol glutaredoxins (Grxs) playing an important role. However, monothiol Grxs are not considered to have major oxidoreductase activity. The present study investigated the redox regulation of yeast Sir2, together with the role and physiological impact of monothiol Grx3/4 as Sir2 thiol-reductases upon stress. S-glutathionylation of Sir2 upon disulfide stress was demonstrated both in vitro and in vivo, and decreased Sir2 deacetylase activity. Physiological levels of nuclear Grx3/4 can reverse the observed post-translational modification. Grx3/4 interacted with Sir2 and reduced it after stress, thereby restoring telomeric silencing activity. Using site-directed mutagenesis, key cysteine residues at the catalytic domain of Sir2 were identified as a target of S-glutathionylation. Mutation of these residues resulted in cells with increased resistance to disulfide stress. We provide new mechanistic insights into Grx3/4 regulation of Sir2 by S-deglutathionylation to increase cell resistance to stress. This finding offers news perspectives on monothiol Grxs in redox signaling, describing Sir2 as a physiological substrate regulated by S-glutathionylation. These results might have a relevant role in understanding aging and age-related diseases.

Oxidative stress has been described as important to Huntington disease (HD) progression. In a previous HD study, we identified several carbonylated proteins, including pyridoxal kinase and antiquitin, both of which are involved in the metabolism of pyridoxal 5´-phosphate (PLP), the active form of vitamin B6. In the present study, pyridoxal kinase levels were quantified and showed to be decreased both in HD patients and a R6/1 mouse model, compared to control samples. A metabolomic analysis was used to analyze metabolites in brain samples of HD patients and R6/1 mice, compared to control samples using mass spectrometry. This technique allowed detection of increased concentrations of pyridoxal, the substrate of pyridoxal kinase. In addition, PLP, the product of the reaction, was decreased in striatum from R6/1 mice. Furthermore, glutamate and cystathionine, both substrates of PLP-dependent enzymes were increased in HD. This reinforces the hypothesis that PLP synthesis is impaired, and could explain some alterations observed in the disease. Together, these results identify PLP as a potential therapeutic agent.