Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis

Aims: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway.

Results: Exposure of HEK 293T cells to hydrogen peroxide (H₂O₂) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H₂O₂. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism.

Conclusions: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.