Nitrated Nuclear Receptor Coactivator 4 Contributes to Ferritinophagy in Hyperhomocysteinemia-Induced Liver Injury

Aims: Hyperhomocysteinemia (HHcy) has been established as a significant risk factor for liver diseases, and its impact on nitrative stress and protein nitration has also been recognized. Nuclear receptor coactivator 4 (NCOA4) is crucial in maintaining iron homeostasis and regulating ferroptosis. However, limited studies are available regarding the post-translational modifications (PTMs) of NCOA4 in liver injury. Results: Mice fed with a diet containing 2.5% methionine and water with 1.8 g/L DL-Hcy were used in this study. The HHcy-induced liver injury phenotype was assessed through serum aspartate aminotransferase/alanine aminotransferase (AST/ALT), hematoxylin and eosin staining, Masson staining, and real-time quantitative PCR. To further analyze the mechanisms, we conducted tandem mass tag proteomics analysis, Perls blue staining, transmission electron microscopy observation, liver iron content determination, malondialdehyde level, autophagic flux analysis, and detection of indicators related to ferritinophagy–ferroptosis pathway. Additionally, the nitrated NCOA4 was enhanced in HHcy stimulation, especially at sites such as Tyr78, Tyr214, Tyr269, Tyr335, and Tyr608. Peroxynitrite decomposition catalyst (PDC) was used to clear peroxynitrite, while nitrated NCOA4 was inhibited, thereby alleviating liver injury caused by HHcy. We established an HHcy mouse model and confirmed liver injury by elevated serum AST/ALT, histopathology, and fibrosis markers. Mechanistically, HHcy led to hepatic iron overload, lipid peroxidation, and ferroptosis. These effects were associated with upregulated autophagy and NCOA4-mediated ferritinophagy. Importantly, we confirmed in vitro that HHcy enhanced the nitration of NCOA4 at specific tyrosine residues (Tyr78, 214, 269, 335, 608). Scavenging peroxynitrite with a decomposition catalyst inhibited NCOA4 nitration, subsequently suppressing ferritinophagy, reducing iron overload and lipid peroxidation, and ultimately alleviating liver injury. Innovation and Conclusion: We find that HHcy induces nitrative modification at Tyr78/214/269/335/608 sites of NCOA4, upregulating ferritinophagy levels, increasing the intracellular labile iron pool, and triggering iron-dependent lipid peroxidation-driven ferroptosis, thereby promoting the occurrence and development of liver injury. These data indicate that inhibiting nitrated NCOA4, thereby curtailing ferritinophagy, represents a potential therapeutic target for the treatment of HHcy-induced liver injury. This study is the first to identify NCOA4 nitration as a critical PTM that amplifies ferritinophagy under conditions of nitrative stress. We pinpoint five specific nitration sites on NCOA4 and establish a novel causal link between HHcy, protein nitration, and ferroptosis-driven liver injury. Inhibiting the nitrated NCOA4 or upstream peroxynitrite formation to restrain pathological ferritinophagy represents a promising therapeutic strategy for HHcy-induced liver injury. Antioxid. Redox Signal. 00, 000–000.