Non-transcriptional regulation of circadian physiology

昼夜节律钟是整合外部时间线索的内源性蛋白质机器， 内部代谢状态，以调节生物体的生理和行为的日常节奏， 所有生命的王国在自然界中，环境时间表使动物的昼夜节律 控制进食时间的生物钟。因此，养分流入可以提供代谢信号， 增强环境信号，促进昼夜生理同步， 代谢和能量利用。解剖昼夜节律振荡器基础的初步努力 它对生命节律的控制集中在转录水平上， 振荡器蛋白是一种转录因子，它们共同控制基因的节律性表达 参与多种细胞过程。最近的研究发现了互补的非转录机制，包括蛋白质翻译后修饰（PTM）， 对昼夜节律的计时至关重要。本项目的总体目标是了解 代谢和环境信号在翻译后整合的机制 水平来调节昼夜生理，更重要的是，当这些 已经进化为合作的信号是冲突的。我们将使用昼夜果蝇模型 为了验证中心假设，即营养流入通过时钟控制的摄食活动， 调节细胞蛋白质的磷酸化和O-连接的N-乙酰葡糖胺化（O-GlcNAc化）之间的相互作用，以调节一天中特定时间的功能。蛋白质O-GlcNAc化对代谢输入高度敏感，并且可能在广泛的代谢过程中起主导作用。 重塑细胞蛋白质功能，绕过基因表达的变化。在目标1中，我们 使用时间限制喂养（TRF）结合靶向代谢组学， 化学酶O-GlcNAc标记以建立进食-禁食周期之间的关系， 营养流入和细胞蛋白质的O-GlcNAc化状态。在目标2中，我们将识别细胞 使用无标记蛋白质组学方法，显示O-GlcNAc酰化和磷酸化之间的日常相互作用的蛋白质。在目标3中，我们将利用可靠的分子和果蝇行为分析来表征时钟蛋白O-GlcNAc化事件的功能。通过解决这三个问题：什么时候，什么，为什么，我们将进一步了解 通过翻译后机制的昼夜生理代谢调节。该项目将 具有广泛的意义，因为蛋白质磷酸化和O-GlcNAc酰化之间的串扰是 广泛的和调节广泛的细胞过程。我们的发现可能会发现新的 治疗目标，以减轻昼夜节律和代谢紊乱。

Circadian clocks are endogenous protein machines that integrate external time cues and internal metabolic states to regulate daily rhythms in physiology and behavior in organisms from all kingdoms of life. In the natural world, environmental zeitgebers enable the animal circadian clock to control timing of food intake. Nutrient influx can therefore provide metabolic signals to reinforce environmental signals, promoting synchrony in circadian physiology to balance metabolism and energy use. Initial efforts to dissect the underpinnings of the circadian oscillator and its control over rhythms of life focused on regulation at the transcriptional level, as the core oscillator proteins are transcription factors that collaborate to govern rhythmic expression of genes involved in diverse cellular processes. More recent studies have uncovered complementary non-transcriptional mechanisms, including protein post-translational modifications (PTMs), that are critical for circadian timekeeping. The overall goal of this project is to understand the mechanisms by which metabolic and environmental signals integrate at the post-translational level to regulate circadian physiology, and more importantly the consequences when these signals that have evolved to cooperate are in conflict. We will use the diurnal Drosophila model to test the central hypothesis that nutrient influx through clock-controlled feeding activity regulates the interplay between phosphorylation and O-linked N-Acetylglucosaminylation (O-GlcNAcylation) of cellular proteins to modulate time-of-day specific functions. Protein O-GlcNAcylation is highly sensitive to metabolic input and may play a dominant role in extensive remodeling of cellular protein functions, bypassing changes in gene expression. In Aim 1, we will use time-restricted feeding (TRF) in combination with targeted metabolomics and chemoenzymatic O-GlcNAc labeling to establish the relationships between feeding-fasting cycle, nutrient influx, and O-GlcNAcylation status of cellular proteins. In Aim 2, we will identify cellular proteins that exhibit daily interplay between O-GlcNAcylation and phosphorylation using label-free proteomic approaches. In Aim 3, we will characterize the function of clock protein O-GlcNAcylation events by utilizing tried-and-true molecular and Drosophila behavioral assays. By addressing the 3 questions: When, What, and Why, we will advance our understanding on metabolic regulation of circadian physiology via post-translational mechanisms. This project will have broad significance as cross-talk between protein phosphorylation and O-GlcNAcylation is extensive and modulates a wide range of cellular processes. Our findings may identify new therapeutic targets to alleviate circadian and metabolic disorders.