Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

Abstract O-GlcNAcylation, the reversible addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues, is a dynamic posttranslational modification that integrates nutrient and stress signals to fine-tune protein function and maintain cellular homeostasis. Although dysregulation of O-GlcNAc signaling is associated with age-related pathologies, its role in physiological aging remains unclear. Here we show that chronologically aged human vascular endothelial cells exhibit reduced O-GlcNAcylation due to altered abundance of enzymes in the hexosamine biosynthesis pathway and O-GlcNAc cycling. Conversely, the attenuation of O-GlcNAcylation achieved by genetic or pharmacological means, induced senescence via canonical p53/p21 CIP1 and p16 INK4a /Rb pathways and impaired key endothelial functions, such as proliferation and angiogenic capacity. Importantly, O-GlcNAcylated substrates identified across the entire proteome in endothelial cells closely aligned with these mechanistic and phenotypic findings. These substrates were enriched in regulators of processes central to senescence and vascular aging, such as genome stability, transcription, cell cycle, and methylation. Among these, we confirmed cell division cycle and apoptosis regulator 1 (CCAR1) as a bona fide O-GlcNAc substrate and demonstrate that its expression and O-GlcNAcylation decline during senescence in endothelial cells. CCAR1 depletion suppressed apoptosis and sensitized cells to oxidative stress-induced DNA damage, whereas restoration of CCAR1 levels mitigated premature and replicative senescence. Together, our findings establish O-GlcNAc signaling as a key regulatory system that integrates metabolic and stress cues to coordinate protein networks controlling genome stability, transcription and cellular stress adaptation, thereby preserving endothelial integrity and restraining senescence onset.