Dynamic O-GlcNAcylation coordinates ferritinophagy and mitophagy to activate ferroptosis

Ferroptosis, a type of regulated necrosis characterized by iron-dependent lipid peroxidation, is implicated in various diseases. Its initiation involves factors like Erastin (cysteine import inhibitor) and RSL3 (GPX4 inhibitor), disrupting redox homeostasis and leading to lipid peroxidation. Multiple metabolic processes, including amino acid, lipid, and iron metabolisms, influence ferroptosis. Redox-active iron fuels lipid peroxidation, and its homeostasis is crucial, involving absorption, storage (by ferritin), and efflux. Ferritin, composed of heavy and light chains, can be degraded via ferritinophagy (mediated by NCOA4), releasing iron and contributing to ferroptosis. While the role of iron in ferroptosis is established, the precise activation mechanisms of ferritinophagy-dependent ferroptosis remain unclear. O-GlcNAcylation, a post-translational modification regulated by OGT and OGA, is a nutrient sensor responding to various stresses. It plays paradoxical roles in diseases, and its involvement in ferroptosis remains unexplored. This study investigates the role of O-GlcNAcylation in sensing ferroptotic stress and coordinating ferritinophagy and mitophagy.

Existing literature extensively details the role of iron and lipid peroxidation in ferroptosis, highlighting the involvement of various metabolic pathways. Studies have shown that inhibiting systems that defend against lipid peroxidation, such as glutathione metabolism, can trigger ferroptosis. The role of iron in ferroptosis is well-established, with studies showing that chelating labile iron prevents ferroptosis. Ferritinophagy, the lysosomal degradation of ferritin to release iron, has emerged as a critical process contributing to ferroptosis. However, the upstream regulatory mechanisms governing ferritinophagy and the interplay between various metabolic pathways in ferroptosis are not fully understood. The literature also highlights the importance of O-GlcNAcylation in various cellular processes and its role as a nutrient sensor, but its involvement in ferroptosis was largely unexplored before this study.

The study utilized various techniques to investigate the role of O-GlcNAcylation in ferroptosis. Immunoblotting was used to analyze O-GlcNAcylation levels following treatment with ferroptosis inducers (RSL3, ML210, iPSP1, Erastin). RNA interference (RNAi) was employed to genetically inhibit GPX4. Flow cytometry was used to quantify O-GlcNAcylation levels and lipid ROS production. Cell viability assays (CCK8 and Trypan blue staining) were performed to assess the impact of O-GlcNAcylation inhibition on cell death. Immunofluorescence microscopy was utilized to examine the effects of O-GlcNAcylation inhibition on cellular morphology and protein localization. Different cell lines (U2OS, HT1080, HUVEC, HT29) were used to ensure the generalizability of the findings. Pharmacological inhibitors of O-GlcNAcylation (OSMI-1, TMG) were used to manipulate O-GlcNAcylation levels.

The study revealed a biphasic change in O-GlcNAcylation levels following ferroptosis induction. Initially, O-GlcNAcylation increased, possibly as a cellular defense mechanism. However, subsequent inhibition of O-GlcNAcylation promoted ferritinophagy, leading to increased labile iron and mitochondrial damage. Inhibition of O-GlcNAcylation also enhanced mitophagy, further contributing to increased labile iron and ferroptosis. Mechanistically, de-O-GlcNAcylation of ferritin heavy chain at serine 179 (S179) facilitated its interaction with NCOA4, the receptor for ferritinophagy, thus promoting iron release and ferroptosis. Inhibition of O-GlcNAcylation significantly increased cell death induced by RSL3, indicating a crucial role of O-GlcNAcylation in regulating ferroptosis sensitivity. The effects were consistent across multiple cell lines, suggesting a conserved mechanism.

This study establishes a crucial link between O-GlcNAcylation and ferroptosis, revealing a previously uncharacterized mechanism by which metabolic stress influences iron homeostasis and mitochondrial function to regulate cell death. The biphasic nature of O-GlcNAcylation suggests a complex interplay between cellular defense mechanisms and the execution of ferroptosis. The findings highlight the importance of O-GlcNAcylation in controlling iron availability and mitochondrial integrity, two key factors in ferroptosis. This work significantly expands our understanding of ferroptosis regulation and offers potential therapeutic targets for diseases where ferroptosis plays a role.

This study demonstrates that dynamic O-GlcNAcylation acts as a critical regulator of ferroptosis by coordinating ferritinophagy and mitophagy. The de-O-GlcNAcylation of ferritin heavy chain at S179 enhances its interaction with NCOA4, leading to labile iron accumulation and ferroptosis. These findings uncover a new regulatory mechanism of ferroptosis and provide potential therapeutic targets for diseases involving this form of cell death. Future research could focus on exploring the specific downstream effectors of O-GlcNAcylation in ferroptosis and investigating the therapeutic potential of modulating O-GlcNAcylation in relevant disease models.

While the study provides compelling evidence for the role of O-GlcNAcylation in ferroptosis, further investigations are needed to fully elucidate the precise molecular mechanisms involved. The study primarily focused on in vitro experiments, and further in vivo studies are required to confirm the findings in a whole organism context. The specific contribution of mitophagy compared to ferritinophagy in the overall process also requires further investigation.