Liu Xianhui, Cai Yao D, Hou Chunyan, Liu Xu, Luo Youcheng, Mendiola Aron Judd P, Xu Xuehan, Luo Yige, Zheng Haiyan, Zhao Caifeng, Chen Ching-Hsuan, Zhang Yong, Xiang Yang K, Ma Junfeng, Chiu Joanna C
Cambridge-Suda Genomic Resource Center, The Fourth Affiliated Hospital, Suzhou Medical College, Soochow University, Suzhou, China.
Department of Entomology and Nematology, University of California, Davis, California, United States of America.
PLoS Biol. 2025 Sep 25;23(9):e3003400. doi: 10.1371/journal.pbio.3003400. eCollection 2025 Sep.
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 observe daily oscillation of global nuclear protein O-GlcNAcylation in the liver of mice subjected to night-restricted feeding (NRF) using label-free global O-GlcNAc proteomics. Additional site-specific O-GlcNAc analysis by tandem mass tag mass spectrometry further supports temporal differences in O-GlcNAcylation by revealing day-night differences. Proteins involved in gene expression are enriched among rhythmically O-GlcNAcylated proteins, suggesting rhythmic O-GlcNAcylation may directly regulate the hepatic transcriptome. We show that rhythmic O-GlcNAcylation can also indirectly modulate nuclear proteins by interacting with phosphorylation. Several proteins harboring O-GlcNAcylation-phosphorylation interplay motif exhibit rhythmic O-GlcNAcylation and phosphorylation. Specifically, we show that O-GlcNAcylation occurs 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 significantly alters O-GlcNAcylation pattern. Whereas global O-GlcNAcylation analysis indicates dampening of global O-GlcNAcylation rhythm in mice fed under DRF, site-specific analysis reveals differential responses of O-GlcNAc sites when timing of food intake is altered. Notably, a substantial number of O-GlcNAcylation sites exhibit inverted day-night profiles when mice are subjected to DRF. This suggests the dysregulation of daily nuclear protein O-GlcNAcylation rhythm may contribute to the disruption in liver transcriptome previously observed in DRF condition. In summary, our results provide new mechanistic insights into metabolic regulation of hepatic transcriptional regulators via interplay between O-GlcNAcylation and phosphorylation and shed light on the deleterious effects of improper mealtimes.
肝脏生物钟和肝脏转录组对进食-禁食节律产生的代谢信号高度敏感。以往的研究已经确定了许多营养敏感信号通路，这些通路可以解读代谢输入以调节节律性肝脏生物学。在此，我们研究了O-连接N-乙酰葡糖胺化（O-GlcNAcylation）这一营养敏感的翻译后修饰（PTM）在介导肝脏节律性生物学代谢调节中的作用。我们使用无标记全局O-GlcNAc蛋白质组学观察了夜间限时喂食（NRF）小鼠肝脏中全局核蛋白O-GlcNAcylation的每日振荡。通过串联质量标签质谱进行的额外位点特异性O-GlcNAc分析通过揭示昼夜差异进一步支持了O-GlcNAcylation的时间差异。参与基因表达的蛋白质在节律性O-GlcNAc化蛋白质中富集，这表明节律性O-GlcNAcylation可能直接调节肝脏转录组。我们表明，节律性O-GlcNAcylation还可以通过与磷酸化相互作用间接调节核蛋白。几种具有O-GlcNAcylation-磷酸化相互作用基序的蛋白质表现出节律性O-GlcNAcylation和磷酸化。具体而言，我们表明O-GlcNAcylation发生在关键昼夜节律转录激活因子昼夜运动输出周期蛋白（CLOCK）的磷酸化降解基序上，从而调节其稳定性和转录输出。最后，我们报告夜间小鼠的日间限时喂食（DRF）显著改变了O-GlcNAcylation模式。虽然全局O-GlcNAcylation分析表明DRF喂养的小鼠中全局O-GlcNAcylation节律减弱，但位点特异性分析揭示了食物摄入时间改变时O-GlcNAc位点的不同反应。值得注意的是，当小鼠接受DRF时，大量O-GlcNAcylation位点呈现昼夜颠倒的分布。这表明每日核蛋白O-GlcNAcylation节律失调可能导致先前在DRF条件下观察到的肝脏转录组破坏。总之，我们的结果为通过O-GlcNAcylation和磷酸化之间的相互作用对肝脏转录调节因子进行代谢调节提供了新的机制见解，并揭示了不当进餐时间的有害影响。
