Cycling of circadian rhythm proteins

昼夜节律蛋白的循环

• 批准号: 7983858
• 负责人: AMITA SEHGAL
• 金额: $ 34.74万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/7983858
• 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; receptor; response; shift work; small molecule; therapy development; tool; tumor growth; ubiquitin-protein ligase

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. PUBLIC HEALTH RELEVANCE: Circadian rhythms pervade all aspects of physiology and behavior, such as sleep-wake, body temperature, hormonal secretions, blood pressure and liver metabolism. Disruption of these rhythms, which may be caused by many conditions including shift work, has been associated with metabolic disorders, sleep problems and even tumor growth (Boivin et al., 2007; Fu et al., 2002; Toh et al., 2001; Xu et al., 2005). An understanding of the molecular mechanisms that drive these rhythms will lead to the development of treatments for rhythm dysfunction.

描述（由申请人提供）：长期目标是了解昼夜节律的分子基础。这些节奏由大多数生物的内源性控制，在许多不同的生理过程中表现出来。时钟功能中断与睡眠障碍以及其他病理（例如肿瘤生长）有关。内源性昼夜节律时钟的分子特性主要是通过果蝇果蝇中的研究来确定的。这些研究表明，时钟由特定基因（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。因此，反馈回路驱动了下游生理成分的循环，从而驱动行为和生理的节奏。但是，尚不清楚以24小时周期性保持这种反馈循环的机制。此外，节奏通常在某些时钟mRNA，有时甚至是时钟蛋白的水平都保持恒定的条件下保持，这表明翻译后调节的关键作用。果蝇时钟的同步也依赖于这种翻译后机制。我们假设必须循环循环以保持时钟功能的反馈活动，并且该活动的循环是由对蛋白质稳定性，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，但尚未确定关键的调节步骤。我们建议使用最近发现/生成的工具来剖析主要自行车果蝇蛋白，周期（PER）和永恒（TIM）的翻译后调节。我们还将研究时钟对光的反应，该响应在控制蛋白质稳定性方面具有基础。具体目的是：（1）确定新型Tim等位基因行为表型的基础机制，我们已经确定了TIM的新型突变，该突变会影响Per和Tim的稳定性和核定位，以及PER的磷酸化。这为我们提供了一种强大的工具来解决核表达的基础机制，并确定亚细胞定位对磷酸化和稳定性的影响； （2）确定新型磷酸化位点在Per和Tim上的功能意义。我们已经通过质谱分析确定了Per和TIM的新型磷酸化位点。我们将研究这些站点在上面列出的过程中的作用； （3）确定TIM对光的反应所需的激酶。通过一个小的分子抑制剂筛选，我们已经确定了tim降解所需的类别的激酶。我们将确定所涉及的特定激酶。 公共卫生相关性：昼夜节律遍及生理和行为的各个方面，例如睡眠，体温，荷尔蒙分泌物，血压和肝代谢。这些节奏的破坏可能是由包括转移工作在内的许多条件引起的，与代谢障碍，睡眠问题甚至肿瘤生长有关（Boivin等，2007; Fu等，2002; Toh等，2001; Xu等，2005）。对驱动这些节奏的分子机制的理解将导致节奏功能障碍的治疗发展。