Reversible glutathionylation of Sir2 by monothiol glutaredoxins Grx3/4 regulates stress resistance

Abstract

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.

Glutaredoxin-1 Deficiency Causes Fatty Liver and Dyslipidemia by Inhibiting Sirtuin-1

“…Activation of SirT1 improved NAFL and conversely hepatic SirT1 deficiency led to steatosis (56). Inhibition of SirT1 activity by reversible GSH adducts has recently been described by our group and was confirmed by other investigators (11,67,71,77). Because NAFL (58) is associated with oxidative stress, increased protein GSH adducts may play an important role in the pathogenesis of steatotic livers.…”

Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

“…An inactivating WP-motif also makes sense from a physiological perspective, because it might allow the stabilization of a modified sensor and/or avoid the accumulation of trapped Grx-SS-protein species in the absence of a resolving cysteine 7,11,13 . For example, ScGrx3 and ScGrx4 both have a WPmotif and were shown to deglutathionylate very slowly the histone deacetylase Sir2 in a redox-dependent signaling cascade 54 .…”

The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

“…For example, numerous proteins have cysteine residues that form intramolecular disulfide bonds or that can undergo a glutathionylation, that is, form a mixed disulfide bond with glutathione in vitro . The disulfide bonds can be reduced again by Grx and such reversible thiol/disulfide exchange reactions are used to capture and identify candidate proteins ( 4 , 163 , 187 , 243 , 247 , 264 , 283 ). For many of these candidates, however, it remains to be clarified whether and how disulfide bond formation and glutathionylation occur under physiological conditions ( 65 ) (see also the Kinetic Competitions for GSSG and GSSR section).…”