Role of Phosphorylation in Determining Circadian Period Length and Temperature Compensation

项目总结 24小时的生理内源性调节循环，称为昼夜节律，使生物体 与地球固有的自转有关。昼夜节律影响许多基本过程的调节， 包括睡眠调节。睡眠由昼夜节律和睡眠平衡相结合来调节 两组分睡眠模型中的机制。这项建议旨在加深我们对睡眠的理解 通过研究昼夜节律的分子机制中仍然存在的关键缺口来进行调控，这样我们就可以 可能了解基于昼夜节律的睡眠障碍的潜在机制。因此，填补这些空白将 使我们能够开发睡眠障碍的治疗方法。昼夜节律在一定程度上是由大约24小时的周期来定义的 长度，以及它们在环境温度(温度)变化中保持一致周期的能力 薪酬-TC)。尽管这些属性是基本的，但关于它们的根本问题仍然存在 机械装置。我们将研究为分子时钟提供动力的反馈回路是如何完成循环的 (决定周期长度)在目标1中，以及激酶和特定的磷酸化事件在TC中的作用 目标2中的机制。目前的证据表明，这两种属性最终都受到 磷酸化。我们假设，确定周期的机制是保守的 脉孢子虫对哺乳动物的影响，因此哺乳动物生物钟的反馈环被关闭 负性成分的过度磷酸化，而不是降解，与我们实验室在脉孢子菌中发现的情况一致。 Aim 1验证了我们的假设，即时钟蛋白PER2的磷酸化状态而不是稳定性决定了 哺乳动物细胞中的周期长度。我们假设TC涉及KEY的精确和动态磷酸化。 酪蛋白激酶I和II的时钟成分。AIM 2使用磷酸蛋白质组学和上位性的组合 用新的脉孢菌菌株进行实验，以建立一个综合的TC模型，然后我们将在 哺乳动物细胞在培养中，再次期待机械保护。总体而言，这个项目将揭示 昼夜节律的基本方面尚未阐明，最终建立了新的模型 包括周期确定和TC。因此，这项工作的顺利完成将通知我们对 在所有以昼夜节律为基础的障碍中，包括家族性晚期睡眠相综合征等障碍 (FASPS)。这项奖学金将使我能够填补我培训中的空白，成为一名独立的学术科学家。 培训将包括参加国际会议、质谱分析和建模课程， 与我的导师、论文委员会和合作者会面，并接受教学技巧培训，其中包括 其他。我将通过部门研讨会利用达特茅斯的严酷环境，并成为 在我的技术培训中，我们优秀的核心设施为我提供了支持。我现在的研究环境将是 邓拉普/洛罗斯实验室几十年来一直是研究昼夜节律的分子基础的中心。

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.