Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.
In 2025, teams from Fudan University, Shanghai Jiao Tong University, etc. published a study in Redox Biology:
This research reveals that the total amount of the glutathione pool regulates protein S-glutathionylation (protein-SSG) to resist ferroptosis, and discovers a new regulatory axis of CHAC1-ARF6-SSG-TFRC.
It provides new targets for the intervention of liver injury related to glutathione depletion and ferroptosis.
Glutathione homeostasis plays a crucial role in regulating various cellular biological functions and in the occurrence and development of diseases.
Glutathione deficiency is closely related to the progression of various human diseases and the aging process.
Among the various functional abnormalities caused by glutathione deficiency, ferroptosis is one of the most significant biological events.
Ferroptosis is a type of regulated cell death driven by lipid peroxidation and dependent on iron ions.
The discovery of ferroptosis can be traced back to the 1950s.
Researchers first identified a form of cell death that triggered specifically by cysteine deficiency, which different from other forms of cell death caused by amino acid deficiencies.
Subsequent decades of research have confirmed that the absence of cysteine leads to depletion of glutathione, ultimately triggering ferroptosis.
Consistent with this mechanism, the first identified ferroptosis inducer, erastin, exerts its effect by inhibiting the cystine/glutamate antiporter SLC7A11.
This inhibitory effect disrupts the synthesis of cysteine-dependent reduced glutathione (GSH), thereby inducing ferroptosis.
Glutathione peroxidase (GPX4) catalyzes the conversion of GSH into oxidized glutathione (GSSG), which is a key antioxidant reaction for removing lipid peroxides.
The GSSG/GSH ratio is widely regarded as a key indicator of the cellular oxidative state.
Excessive oxidative stress is considered an important driving factor for ferroptosis.
In fact, the intracellular GSSG level not only regulated by the redox state but also influenced by the availability of its precursor substrate, glutathione.
In the iron death induced by glutathione depletion in various models, its commonly observed that the total glutathione pool (including GSH and GSSG) significantly decreases.
This suggests that the imbalance in the GSSG/GSH ratio alone may not an adequate explanation for the iron death induced by glutathione depletion.
Protein S-glutathionylation (protein-SSG) is a reversible oxidative post-translational modification that forms a disulfide bond between glutathione and the cysteine residues of the target protein.
Recent studies have shown that it plays a crucial role in regulating enzyme activity, subcellular localization, protein interactions, and stability, thereby influencing the occurrence and progression of various diseases.
The sulfhydryl groups of proteins can first oxidized by reactive oxygen species (ROS) to higher oxidation states such as sulfurous acid, or modified by reactive nitrogen to S-nitrosoglutathione.
Subsequently, they react with GSH to form S-glutathionylation.
GSSG can also directly modify proteins.
When oxidative stress alleviated, protein-SSG can revert and restore to its protein sulfhydryl form.
ROS functions as an important signaling molecule at the cellular level and performs physiological functions, but excessive accumulation leads to oxidative stress.
Protein-SSG is closely related to the oxidative state of cells.
During the ferroptosis process induced by glutathione depletion, the role of protein-SSG remains unclear.
Protein S - glutathionylation can occur spontaneously in the presence of ROS and is closely related to the availability of glutathione.
Both GSH and GSSG can serve as substrates for this modification.
The maintenance of GSH homeostasis depends on the coordinated regulation of its synthesis, transport, excretion, peroxidase (GPX4), reductase, consumption and degradation.
CHAC1 is a key enzyme that catalyzes the degradation of intracellular GSH.
Inactivation of CHAC1 can effectively maintain GSH levels in mouse models.
The important point that in ferroptosis induced by GSH depletion, CHAC1 significantly upregulated and widely regarded as a reliable marker of ferroptosis.
It is still unclear how the upregulation of CHAC1 affects ferroptosis.
Whether CHAC1 regulates the glutathione pool (GSH + GSSG) and collaborates with protein-SSG to affect iron death induced by glutathione depletion is a scientific question that urgently needs to answered.
In ferroptosis, iron catalyzes lipid peroxidation through the Fenton reaction.
Transferrin receptor (TFRC) is the main iron transporter in cells.
Due to its aggregation on the cell membrane, it has recently been identified as a biomarker for ferroptosis.
Inhibiting the expression of TFRC can alleviate ferroptosis and significantly alleviate liver ischemia/reperfusion injury in the older people.
On the contrary, the increase in TFRC levels would exacerbate ferroptosis in Coxsackievirus B3 infections.
Transforming growth factor β increases intracellular Fe²⁺ levels by upregulating TFRC, thereby activating fibroblasts.
The absence of TFRC in fibroblasts can inhibit bleomycin-induced pulmonary fibrosis.
ADP - Ribosylation Factor 6 (ARF6) regulates endocytosis mediated by clathrin, endocytosis not mediated by clathrin, and endosomal trafficking.
A typical case of receptor-mediated endocytosis through the action of clathrin is the internalization and recycling of the transferrin (TF)/transferrin receptor (TFRC) complex.
This study confirmed that the upregulation of CHAC1 would exacerbate the ferroptosis induced by glutathione depletion, and it identified that CHAC1 an important regulatory factor of protein-SSG in both the ferroptosis induced by erastin in various cell lines in vitro and the liver cell ferroptosis model triggered by acetaminophen (APAP) in vivo.
Using quantitative redox proteomics, we identified 482 proteins that undergo glutathionylation under steady-state conditions.
Among these, the modification levels of 221 proteins regulated by CHAC1 in ferroptosis.
As an important example, we found that ARF6 regulated by S-glutathionylation during ferroptosis induced by glutathione depletion.
The S-glutathionylation of ARF6 can regulate the expression of TFRC and the uptake of transferrin, and it exerts an inhibitory effect on the process of ferroptosis.
This study provides direct evidence for the availability of the glutathione pool, which affects protein S-glutathionylation, regulates protein function, and thereby influences the process of ferroptosis.
It opens up a new direction for understanding the mechanism of cell ferroptosis induced by glutathione depletion.
CHAC1 upregulation → depletion of the total glutathione pool (GSH + GSSG) → reduction of ARF6-Cys90 S - glutathionylation → decreased ARF6 lysosomal localization → increase in cell membrane TFRC → increased transferrin uptake → increased Fe²⁺ → increased lipid peroxidation → increased ferroptosis → liver injury / tumor cell death