Site-specific TMT O-GlcNAc proteomics

ABSTRACT: The liver circadian clock and hepatic transcriptome are highly responsive to metabolic signals generated from feeding-fasting rhythm. Previous studies have identified a number of nutrient-sensitive signaling pathways that could interpret metabolic input to regulate rhythmic hepatic biology. Here, we investigated the role of O-GlcNAcylation, a nutrient-sensitive post-translational modification (PTM) in mediating metabolic regulation of rhythmic biology in the liver. We observed daily oscillation of global nuclear protein O-GlcNAcylation in the liver of mice subjected to night-restricted feeding (NRF). Among 449 O-GlcNAcylated proteins we identified, 64 proteins are rhythmically O-GlcNAcylated over a 24-hour day-night cycle. Proteins involved in gene expression were enriched among rhythmically O-GlcNAcylated nuclear proteins, suggesting rhythmic O-GlcNAcylation may directly shape the daily rhythmicity of the hepatic transcriptome. We also identified xxx O-GlcNAcylation sites, demonstrating day-night differences of site-specific O-GlcNAcylation. Furthermore, we showed that rhythmic O-GlcNAcylation can also indirectly modulate the hepatic transcriptome by interacting with phosphorylation. Specifically, several proteins harboring O-GlcNAcylation-phosphorylation interplay motif exhibit rhythmic O-GlcNAcylation and phosphorylation. We show that O-GlcNAcylation occur at a phospho-degron of a key circadian transcriptional activator, circadian locomotor output cycles kaput (CLOCK), thus regulating its stability and transcriptional output. Finally, we report that day-restricted feeding (DRF) in the nocturnal mouse dampens O-GlcNAcylation rhythm. This suggests the dysregulation of daily nuclear protein O-GlcNAcylation rhythm could partially contribute to the disruption in liver transcriptomic rhythm previously observed in DRF condition, despite not the primary driver. In summary, our results provide new mechanistic insights into metabolic regulation of daily hepatic transcriptomic rhythm via interplay between O-GlcNAcylation and phosphorylation and shed light on the deleterious effects of improper mealtimes.

INSTRUMENT(S): nanoUPLC-MS/MS

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Joanna Chiu  

PROVIDER: MSV000097856 | MassIVE | Tue May 13 13:24:00 BST 2025

REPOSITORIES: MassIVE