Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion

Ferroptosis is one of the most critical biological consequences of glutathione depletion. Excessive oxidative stress, indicated by an elevated oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, is recognized as a key driver of ferroptosis. However, in glutathione depletion-induced ferroptosis, a marked decrease in total glutathione levels (including both GSH and GSSG) is frequently observed, yet its significance remains understudied. Protein S-glutathionylation (protein-SSG) levels are closely linked to the redox state and cellular glutathione pools including GSH and GSSG. To date, the role of protein-SSG during cell ferroptosis induced by glutathione depletion remains poorly understood. Here, we demonstrated that upregulation of CHAC1, a glutathione-degrading enzyme, acted as a key regulator of protein-SSG formation and exacerbated glutathione depletion-induced ferroptosis. This effect was observed in both in vitro and in vivo models, including erastin-induced ferroptosis across multiple cell lines and acetaminophen overdose-triggered ferroptosis in hepatocytes. Deficiency of CHAC1 resulted in increased glutathione pools, enhanced protein-SSG, improved liver function, and attenuation of hepatocyte ferroptosis upon acetaminophen challenge. These protective effects were reversed by CHAC1 overexpression. Using quantitative redox proteomics, we identified glutathione pool-sensitive S-glutathionylated proteins. As an important example, we discovered that ADP-ribosylation factor 6 (ARF6) was regulated by S-glutathionylation during glutathione depletion-induced ferroptosis. Our findings revealed that CHAC1 upregulation reduced the S-glutathionylation of ARF6, resulting in decreased ARF6 levels in lysosomes. This, in turn, enhanced the localization of the transferrin receptor (TFRC) on the cell membrane and increased transferrin uptake, ultimately compromising the protective role of ARF6 in ferroptosis induced by glutathione depletion. Targeting TFRC using GalNAc-siTfrc mitigated acetaminophen-induced liver injury in vivo. In conclusion, our study provide evidence that availability of glutathione pools affects protein S-glutathionylation and regulates protein functions to influence the process of ferroptosis, which opens an avenue to understanding the cell ferroptosis induced by glutathione depletion.