The role of DBT and NEMO-dependent phosphoproteome in regulating animal clockwork

DBT 和 NEMO 依赖性磷酸蛋白质组在调节动物时钟中的作用

• 批准号: 9132814
• 负责人: JOANNA Chungyen CHIU
• 金额: $ 27.44万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9132814
• 关键词: Address; Advanced Sleep Phase Syndrome; Affect; Affinity Chromatography; Animals; Automobile Driving; Behavior; Biochemical; Biological Assay; Biology; Circadian Rhythms; Clock protein; Complex; DNA Binding; Defect; Development; Disease; Drosophila Proteins; Drosophila genus; Drosophila melanogaster; Event; F-Box Proteins; Generations; Goals; Health; Human; Laser Scanning Confocal Microscopy; Life Cycle Stages; Link; Maps; Mass Spectrum Analysis; Metabolism; Modeling; Modification; Monitor; Mutation; Neurons; Phase; Phospho-Specific Antibodies; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Population; Property; Protein Phosphatase 2A Regulatory Subunit PR53; Proteomics; Recruitment Activity; Regulation; Role; Serine; Site; Sleep Disorders; Speed; Testing; Time; Transcription Repressor/Corepressor; Transgenic Organisms; Ubiquitination; base; casein kinase I; drug development; fly; high throughput screening; in vivo; large scale production; multicatalytic endopeptidase complex; new technology; programs; protein protein interaction; public health relevance; small molecule; small molecule therapeutics; time use; tissue culture; transcription factor; transcriptome

DESCRIPTION (provided by applicant): The overall goal of this proposal is to better understand the multiple roles of protein phosphorylation in regulating circadian rhythms by focusing on PERIOD (PER), a core transcriptional repressor of the circadian transcriptome and the key biochemical timer that underlies animal circadian time-keeping mechanisms. In animal clocks, de novo synthesized PER goes through a dynamic multi-site phosphorylation program that is dependent on the activities of a number of kinases and phosphatases. Through evolutionary fine-tuning, completion of this phosphorylation cycle requires the duration of a circadian day, thereby closely linking PER phosphorylation program to the speed of the clock. The phase-specific phosphorylation program of PER provides sophisticated time-of-day specific modulations to its functional properties, including stability, subcellular localization, and transcriptional activity, thereby controlling the time and duration for which PER functions as a repressor of the circadian transcriptome. We have previously performed mechanistic studies on specific PER phosphorylation sites that are dependent on DOUBLETIME (DBT) and NEMO kinase activities, and found that they are integral part of a phosphorylation circuitry that sets te pace of the clock. In order to fully comprehend the role of DBT and NEMO-dependent PER phosphorylation in circadian biology, we are proposing to use Drosophila melanogaster as a model to address three main questions. (i) What are the sites that are modified by DBT and NEMO? (ii) What is the temporal progression of the phosphorylation program? (iii) What is the function of specific phosphorylation events? Our specific aims are to (1) map DBT- and NEMO-dependent PER phosphorylation sites using quantitative mass spectrometry; (2) decipher the temporal progression of the PER phosphorylation program in whole animals using phosphospecific antibodies in combination with laser scanning confocal microscopy; and (3) assay changes in PER interactome with respect to kinase activities and time using AP-MS (Affinity Purification and Mass Spectrometry) in order to generate new hypotheses regarding temporal changes in PER functional properties. By integrating the results from our three aims, we will understand how dynamic phase-specific phosphorylation of Drosophila PER progresses throughout the circadian day and how specific PER phosphorylation states impacts its role in circadian time-keeping. Results from our studies could pave the way for development of small molecule therapeutics and have profound impact on treatment of human disorders associated with phosphorylation defects, e.g. Familial Advanced Sleep Phase Syndrome (FASPS).

描述（由申请人提供）：本提案的总体目标是通过关注 PERIOD (PER)，昼夜节律转录组的核心转录抑制因子和其背后的关键生化定时器，更好地了解蛋白质磷酸化在调节昼夜节律中的多重作用。动物昼夜节律计时机制。在动物钟中，从头合成的 PER 会经历动态多位点磷酸化程序，该程序依赖于许多激酶和磷酸酶的活性。通过进化微调，完成这个磷酸化周期需要一个昼夜节律日的持续时间，从而将PER磷酸化程序与生物钟的速度紧密联系起来。 PER 的阶段特异性磷酸化程序为其功能特性（包括稳定性、亚细胞定位和转录活性）提供了复杂的一天中时间特异性调节，从而控制 PER 作为昼夜节律转录组阻遏物发挥作用的时间和持续时间。 我们之前对依赖于 DOUBLETIME (DBT) 和 NEMO 激酶活性的特定 PER 磷酸化位点进行了机制研究，发现它们是决定时钟节奏的磷酸化电路的组成部分。为了充分理解 DBT 和 NEMO 依赖性 PER 磷酸化在昼夜节律生物学中的作用，我们建议使用果蝇作为模型来解决三个主要问题。 (i) DBT 和 NEMO 修改了哪些位点？ (ii) 磷酸化程序的时间进程是怎样的？ (iii) 特定磷酸化事件的功能是什么？我们的具体目标是（1）使用定量质谱法绘制 DBT 和 NEMO 依赖的 PER 磷酸化位点； (2) 使用磷酸特异性抗体结合激光扫描共聚焦显微镜破译整个动物中 PER 磷酸化程序的时间进程； (3) 使用 AP-MS（亲和纯化和质谱）分析 PER 相互作用组相对于激酶活性和时间的变化，以产生关于 PER 功能特性的时间变化的新假设。通过整合我们三个目标的结果，我们将了解果蝇 PER 的动态阶段特异性磷酸化在整个昼夜节律中如何进展，以及特定的 PER 磷酸化状态如何影响其在昼夜节律计时中的作用。我们的研究结果可以为小分子疗法的开发铺平道路，并对与磷酸化缺陷相关的人类疾病的治疗产生深远的影响，例如，家族性高级睡眠阶段综合症（FASPS）。