Glutathiolation Triggers Proteins for Degradation by the Ubiquitin- Proteasome Pathway.

Background: Glutathione is a small antioxidant peptide in cells and it plays an important role in maintaining a reducing intracellular environment. Glutathione is also involved in the dynamic regulation of specific protein functions by reversible glutathiolation of certain proteins in response to oxidative stress. Objective: The purpose of this work is to mechanistically investigate the effects of glutathiolation on the susceptibility of proteins to degradation by the ubiquitin-proteasome pathway (UPP). Methods and Results: The data show that gamma C-crystallin and carbonic anhydrase III were barely degraded by the UPP without modifications, but both were rapidly degraded by the UPP after glutathiolation. Modifications of sulfhydryls by other thiol-modification reagents, such as iodoacetamide, also increased the degradation of gamma C-crystallin, but not as effectively as glutathiolation. Biophysical analysis showed that glutathiolation caused reversible conformational changes of these proteins, including a significant increase in protein surface hydrophobicity and a decrease in thermal stability. The modified protein regained its native conformation and its resistance to degradation upon removal of the glutathione moiety. A cataract-causing T5P mutant gamma C-crystallin shares many biophysical characteristics as glutathiolated gamma C-crystallin, including increased surface hydrophobicity and decreased thermal stability. T5P mutant gamma C-crystallin was also rapidly degraded. Comparison of the conformational changes and the susceptibility to degradation of glutathiolated gamma C-crystallin with other forms of modified gamma C-crystallin suggests that the glutathiolation-induced exposure of hydrophobic patches, rather than the modification per se, serves as the signal for degradation by the UPP. Consistent with this hypothesis, masking the surface hydrophobicity of glutathiolated and T5P mutant gamma C-crystallins significantly reduced their susceptibility to degradation by the UPP. Conclusion: This work demonstrates that glutathiolation is a novel mechanism for the UPP to recognize substrates in response to oxidative stress.