Role of Phosphorylation in Determining Circadian Period Length and Temperature Compensation

磷酸化在确定昼夜节律长度和温度补偿中的作用

• 批准号: 10678253
• 负责人: Elizabeth-Lauren Stevenson
• 金额: $ 4.77万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-16 至 2026-03-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678253
• 关键词: ARNTL gene; Address; Advanced Sleep Phase Syndrome; Animals; Behavior; Biological; Body Temperature; CSNK2A1 gene; Cardiovascular system; Catalytic Domain; Cells; Circadian Dysregulation; Circadian Rhythms; Clock protein; Compensation; Core Facility; Data; Defect; Development; Disease; Educational process of instructing; Elements; Environment; Etiology; Event; Feedback; Fellowship; Genetic; Genetic Epistasis; Genetic Transcription; Health; Hour; International; Knock-out; Laboratory Finding; Length; Malignant Neoplasms; Mammalian Cell; Mammals; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Modeling; Molecular; Mutation; Neurospora; Neurospora crassa; Organism; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Physiology; Planet Earth; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Proteins; Regulation; Reporter; Research; Role; Rotation; Scientist; Site; Sleep; Sleep Disorders; Sleep Disorders Therapy; Sleep disturbances; Speed; System; Techniques; Temperature; Testing; Time; Tissues; Training; Translations; Work; arm; beta-Transducin Repeat-Containing Proteins; casein kinase I; circadian; circadian pacemaker; circadian regulation; experimental study; forward genetics; fungus; improved; insight; kinase inhibitor; meetings; mimetics; model organism; molecular clock; mutant; novel; novel therapeutics; phosphoproteomics; sleep regulation; symposium; therapy development; ubiquitin ligase

PROJECT SUMMARY The 24-hour cycles of endogenous regulation of physiology, known as circadian rhythms, align organisms to the inherent rotation of the earth. Circadian rhythms influence the regulation of many essential processes, including sleep regulation. Sleep is regulated by circadian rhythms in combination with sleep homeostatic mechanisms in the two-component sleep model. This proposal aims to further our understanding of sleep regulation by investigating key gaps that remain in the molecular mechanism of circadian rhythms, so that we may understand the underlying mechanisms of circadian-based sleep disorders. Filling these gaps will therefore enable us to develop therapies for sleep disorders. Circadian rhythms are defined in part by a ~24-hour period length, and their ability to maintain a consistent period across changes in ambient temperature (temperature compensation – TC). Despite the fundamentality of these properties, questions remain regarding their underlying mechanisms. We will investigate how the feedback loop powering the molecular clock completes a cycle (determining period length) in Aim 1, and the role for kinases and specific phosphorylation events in the TC mechanism in Aim 2. Current evidence suggests that both of these properties are ultimately regulated by phosphorylation. We hypothesize that the mechanism underlying period determination is conserved from Neurospora to mammals, and hence the feedback loop of the mammalian clock is closed by hyperphosphorylation of negative elements, rather than degradation, matching what our lab found in Neurospora. Aim 1 tests our hypothesis that phosphorylation status and not stability of the clock protein PER2 determines period length in mammalian cells. We hypothesize that TC involves precise and dynamic phosphorylation of key clock components by Casein Kinase I and II. Aim 2 uses a combination of phosphoproteomics and epistasis experiments with novel Neurospora strains to establish an integrative TC model that we will then test in mammalian cells in culture, again expecting mechanistic conservation. Overall, this project will shed light on fundamental aspects of circadian rhythms yet to be elucidated, ultimately establishing new models for both period determination and TC. Therefore, successful completion of this work will inform the understanding of all circadian-based disorders, including disorders such as Familial Advanced Sleep Phase Syndrome (FASPS). This fellowship will enable me to fill gaps in my training to become an independent academic scientist. Training will include participation in international conferences, mass spectrometry and modeling courses, meetings with my advisors, thesis committee, and collaborators, and training in teaching techniques, among others. I will take advantage of the rigorous environment at Dartmouth through departmental seminars and be supported in my technical training by our excellent core facilities. My immediate research environment will be the Dunlap/Loros lab that has for decades been a center for studying the molecular basis of circadian rhythms.

项目摘要 内源性生理学调节的24小时循环，称为昼夜节律，对齐生物 继承地球的旋转。昼夜节律影响许多基本过程的调节， 包括睡眠调节。睡眠由昼夜节律与睡眠体内平衡相结合来调节 两组分模型中的机制。该建议旨在进一步了解我们对睡眠的理解 通过研究昼夜节律分子机制中保留的关键差距来调节，以便我们 可能了解基于昼夜节律的睡眠障碍的基本机制。因此，填补这些空白会 使我们能够开发睡眠障碍的疗法。昼夜节律的部分定义了约24小时 长度及其在周围温度变化中保持一致周期的能力（温度 补偿 - TC）。尽管这些属性的基本依据，但关于其基础的问题仍然存在 机制。我们将调查反馈回路如何为分子时钟提供动力 （确定周期长度）在AIM 1中，以及TC中激酶和特定磷酸化事件的作用 目标2中的机制。当前的证据表明，这两种特性最终都受到 磷酸化。我们假设从 神经孢子至哺乳动物，因此，哺乳动物时钟的反馈回路被关闭 负元素的高磷酸化而不是降解，与我们的实验室在Neurospora中发现的相匹配。 AIM 1检验了我们的假设，即时钟蛋白PER2的磷酸化状态而不是稳定性确定 哺乳动物细胞的周期长度。我们假设TC涉及钥匙的精确和动态磷酸化 酪蛋白激酶I和II的时钟组件。 AIM 2结合了磷蛋白质组学和上毒 使用新型Neurospora菌株进行实验，以建立一个集成的TC模型，然后我们将在该模型中进行测试 培养中的哺乳动物细胞，再次期望机械保存。总体而言，这个项目将阐明 昼夜节律的基本方面尚未阐明，最终建立了新的模型 两个时期的确定和TC。因此，这项工作的成功完成将为理解提供信息 在所有基于昼夜节律的疾病中，包括家族晚期睡眠期综合征等疾病 （fasps）。这项奖学金将使我能够在培训中填补空白，以成为一名独立的学术科学家。 培训将包括参加国际会议，质谱和建模课程， 与我的顾问，论文委员会和合作者会议，以及教学技术的培训 其他的。我将通过部门半手来利用达特茅斯的严格环境，成为 由我们出色的核心设施支持我的技术培训。我的直接研究环境将是 数十年来的Dunlap/Loros实验室是研究昼夜节律分子基础的中心。