Non-transcriptional regulation of circadian physiology

生物钟是一种内源性蛋白质机器，它整合了外部时间线索和 调节生物体生理和行为的日常节律的内部代谢状态 所有的生命王国。在自然界中，环境诱因使动物的昼夜节律 时钟控制进食的时间。因此，营养物质的流入可以提供代谢信号 强化环境信号，促进昼夜生理的同步性达到平衡 新陈代谢和能量使用。剖析昼夜节律振荡器的基础的初步努力 它对生命节奏的控制集中在转录水平的调控上，这是核心 振荡器蛋白是一种转录因子，它们共同控制基因的有节奏的表达。 参与不同的细胞过程。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)，即 对于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 调节昼夜节律生理的水平，更重要的是，当这些 进化为合作的信号是相互冲突的。我们将使用昼夜果蝇模型 为了验证中心假设，即营养物质通过时钟控制的摄食活动而流入 调节细胞蛋白质的磷酸化和O-连接的N-乙酰氨基葡萄糖基化(O-GlcN酰化)之间的相互作用，以调节特定的时间功能。蛋白质O-GlcN酰化对代谢输入高度敏感，可能在广泛的 重塑细胞蛋白质功能，绕过基因表达的变化。在目标1中，我们将 将限时喂养(TRF)与靶向代谢组学相结合 化学酶标记O-GlcNAc建立喂食-禁食循环， 营养物质内流和细胞蛋白的O-GlcN酰化状态。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在O-GlcN酰化和磷酸化之间每日相互作用的蛋白质。在目标3中，我们将利用久经考验的分子和果蝇行为分析来表征时钟蛋白O-GlcN酰化事件的功能。通过回答以下三个问题：何时、什么和为什么，我们将推进我们对 通过翻译后机制对昼夜节律生理的代谢调节。这个项目将 具有广泛的意义，因为蛋白质磷酸化和O-GlcN酰化之间的串扰是 广泛存在，并调节广泛的细胞过程。我们的发现可能会发现新的 治疗目标是缓解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.