TRAF4-mediated PRMT5 ubiquitination destabilizes GPX4 and promotes endothelial ferroptosis in atherosclerosis

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is a RING-type E3 ubiquitin ligase linked to vascular inflammation, but whether it drives endothelial ferroptosis in atherosclerosis (AS) remains unclear. TRAF4 expression was assessed in GEO dataset GSE166780, ApoE-/- mice fed a high-fat diet for 4 months, and oxidized low-density lipoprotein (ox-LDL)-treated human coronary artery endothelial cells (HCAECs; 50 μg/mL, 24 h). In vivo, mice received tail-vein delivery of shTRAF4 vector. Plaque burden, endothelial injury, inflammatory responses, and ferroptosis were evaluated by histology, qRT-PCR, western blotting, immunofluorescence, reactive oxygen species (ROS) staining, and biochemical assays. TRAF4-PRMT5 and PRMT5-GPX4 interactions were examined by co-immunoprecipitation and GST pull-down. PRMT5 ubiquitination and protein stability were analyzed using MG132 and cycloheximide assays, and rescue experiments were performed by co-silencing TRAF4 and PRMT5. Our results showed that TRAF4 was upregulated in atherosclerotic tissues, the public dataset, and ox-LDL-treated HCAECs. TRAF4 knockdown reduced plaque burden, VCAM-1 expression, serum lipids and inflammatory cytokines, and attenuated ROS accumulation, ferrous iron (Fe²⁺) overload, lipid peroxidation, and Glutathione peroxidase 4 (GPX4) loss in vivo. In HCAECs, TRAF4 silencing improved viability, nitric oxide production, and mitochondrial function while reducing apoptosis, monocyte adhesion, inflammatory cytokine release, and ferroptosis. Mechanistically, TRAF4 directly bound PRMT5 and promoted its K48-linked ubiquitin-dependent degradation. CD31/TRAF4 co-staining confirmed endothelial TRAF4 enrichment in atherosclerotic tissue. PRMT5 bound GPX4, enhanced GPX4 methylation, and stabilized GPX4 protein. PRMT5 knockdown partially reversed the anti-ferroptotic effects of TRAF4 deficiency. Overall, TRAF4 promotes endothelial ferroptosis and AS progression by driving PRMT5 ubiquitination and destabilizing GPX4. The TRAF4-PRMT5-GPX4 axis may represent a therapeutic target in AS.