Nacetyllcysteine Averts Ferroptosis By Fostering Glutathione Peroxidase 4 Jiashuo Zheng 郑嘉烁 Weijia Zhang 张唯佳 Junya Ito Bernhard Henkelmann Chenxi Xu 徐晨曦 Eikan Mishima Marcus Conrad by Jiashuo Zheng (郑嘉烁) & Weijia Zhang (张唯佳) & Junya Ito & Bernhard Henkelmann & Chenxi Xu (徐晨曦) & Eikan Mishima & Marcus Conrad 101016/JCHEMBIOL202504002 instant download after payment.

SIGNIFICANCE Ferroptosis, a distinct form of cell death, has garnered significant attention as both a potential strategy for eradicating cancer cells and a target for therapeutic inhibition in various diseases. Among therapidly expanding body of research on ferroptosis, N-acetyl-L-cysteine (NAC) is frequently employed as a ferroptosis inhibitor. In this study, we investigate its underlying mechanism and find that while NAC primarilyfunctions as a prodrug for cysteine, it may also directly counteract ferroptosis by serving as a reducing substrate for GPX4, the key regulator of ferroptosis. Furthermore, we demonstrate that GPX4 can utilize a widerange of reducing compounds, such as β-mercaptoethanol, as direct reducing substrates. This study enhances our understanding of NAC as a ferroptosis inhibitor and reveals that GPX4 may employ alternativereducing substrates to mitigate ferroptosis, particularly under glutathione-deficient conditions—an important consideration for future research.SUMMARYN-acetyl-L-cysteine (NAC) is a medication andawidely usedantioxidantin celldeath research. Despite its somewhat obscure mechanism of action, its role in inhibiting ferroptosis is gaining increasing recognition. In thisstudy, we demonstrate that NAC treatment rapidly replenishes the intracellular cysteine pool, reinforcing itsfunction as a prodrug for cysteine. Interestingly, its enantiomer, N-acetyl-D-cysteine (D-NAC), which cannotbe converted into cysteine, also exhibits a strong anti-ferroptotic effect. We further clarify that NAC, D-NAC,and cysteine all act as direct reducing substrates for GPX4, counteracting lipid peroxidation. Consequently,onlyGPX4—ratherthan system xc− ,glutathione biosynthesis, orferroptosis suppressor protein 1—is necessaryfor NAC and D-NAC to prevent ferroptosis. Additionally, we identify a broad range of reducing substrates forGPX4 in vitro, including β-mercaptoethanol. These findings provide new insights into the mechanisms underlying the protective effects of NAC and other potential GPX4-reducing substrates against ferroptosis.