Cycling of circadian rhythm proteins

昼夜节律蛋白的循环

基本信息

批准号：
8461162
负责人：
AMITA SEHGAL
金额：
$ 33.51万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8461162
关键词：
Activity Cycles Address Affect Alleles Amino Acids Animal Behavior Behavior Behavioral Biological Assay Blood Pressure Body Temperature Cell Culture Techniques Cell Nucleus Circadian Rhythms Clock protein Cultured Cells Cytoplasm Defect Drosophila genus Drosophila melanogaster Drosophila period protein Event Family Feedback Functional disorder Genes Genetic Screening Genetic Transcription Goals Hormonal Hour Kinetics Lead Libraries Light Link Mass Spectrum Analysis Messenger RNA Metabolic Diseases Modification Molecular Mutate Mutation Nature Nuclear Organism Pathology Periodicity Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Physiological Physiological Processes Physiology Point Mutation Post-Translational Regulation Process Protein Kinase C Protein Tyrosine Kinase Protein phosphatase Proteins Regulation Rest Role Secondary to Site Sleep Sleep Disorders Testing Threonine Time Work base brain cell casein kinase II circadian pacemaker fly genetic analysis inhibitor/antagonist interest kinase inhibitor liver metabolism mutant novel public health relevance receptor response shift work small molecule therapy development tool tumor growth ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called "clock genes") whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock's response to light, which has its basis in the control of protein stability. Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability; (2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above; (3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.

描述（由申请人提供）：长期目标是了解昼夜节律（~24 小时）节律的分子基础。这些节律由大多数生物体的内源性时钟控制，并表现在许多不同的生理过程中。生物钟功能紊乱与睡眠障碍以及肿瘤生长等其他疾病有关。内源生物钟的分子性质主要是通过对果蝇（Drosophila melanogaster）进行的研究来确定的。这些研究表明，生物钟由特定基因（所谓的“时钟基因”）组成，其蛋白质产物在一天中的特定时间调节其自身 mRNA 的合成。由此产生的反馈回路驱动下游生理成分的循环，进而驱动行为和生理的节律。然而，维持这种 24 小时周期反馈循环的机制尚不清楚。此外，节律通常在某些时钟 mRNA（有时甚至时钟蛋白）水平保持恒定的条件下得以维持，这表明翻译后调节的关键作用。果蝇时钟与光的同步也依赖于这种翻译后机制。我们假设时钟蛋白的反馈活性必须循环才能维持时钟功能，并且这种活性的循环是由蛋白质稳定性、核表达和抑制转录能力的时间控制驱动的。时钟蛋白的这些特征在很大程度上受到磷酸化的影响，但关键的调控步骤尚未确定。我们建议使用我们最近发现/生成的工具来剖析主要循环果蝇蛋白、周期（PER）和永恒（TIM）的翻译后调节。我们还将研究生物钟对光的响应，这在控制蛋白质稳定性方面有其基础。具体目标是： (1) 确定新 tim 等位基因行为表型的机制我们已经鉴定了 tim 的新突变，它影响 PER 和 TIM 的稳定性和核定位以及 PER 的磷酸化。这为我们提供了一个强大的工具来解决核表达的机制，并确定亚细胞定位对磷酸化和稳定性的影响； (2) 鉴定PER和TIM上新磷酸化位点的功能意义。我们通过质谱分析确定了 PER 和 TIM 上的新磷酸化位点。我们将调查这些站点在上述流程中的作用； (3) 确定 TIM 光响应所需的激酶。通过小分子抑制剂筛选，我们确定了光降解 TIM 所需的激酶类别。我们将确定所涉及的特定激酶。