Determining the molecular details of the PER2-CK1 delta interaction in the mammalian circadian clock

项目总结 昼夜节律支配着大多数行为和生理过程，以与24小时太阳日相一致。 昼夜节律紊乱会增加患抑郁症、心血管疾病和癌症的风险。这个 蛋白质周期2(Protein Period 2，PER2)位于哺乳动物分子时钟的中心，PER2的水平密切相关 通过酪蛋白激酶1增量(CK1)的磷酸化来调节。一个磷酸化位点的突变 使PER2不稳定，缩短昼夜节律，并导致家族性晚期睡眠相(FASP)综合征 人类。此外，CK1对PER2的磷酸化可能是温度补偿的基础，这 允许时钟产生与温度无关的~24小时计时。CK1基因的分子细节 与PER2相互作用以促进这种稳定的磷酸化，以及这种相互作用如何有助于 人们对温度补偿知之甚少。我研究的长期目标​​是为了理解 PER2的CK1磷酸化如何调节哺乳动物的生物钟。这项提案的重点是 确定CK1如何与PER2相互作用形成稳定的化学计量复合体，以及它如何对 温度补偿。My​Central​​假说​是哺乳动物的温度补偿 生物钟是通过CK1的动力学变化来实现的，这些变化影响了它形成稳定复合体的能力 与PER2和/或使FASP区域磷酸化。这个项目的第一个目标是确定分子 促进复合体形成的PER2-CK1相互作用的细节。使用生物物理技术，我将 一种新的破坏PER2-的小分子抑制剂的络合物和结合的结构表征 CK1相互作用。该项目的第二个目标是确定改变的蛋白质动力学如何有助于 时钟中的温度补偿。动力学可能在几个时间尺度上发生，这使得 利用溶液核磁共振波谱进行研究的合适靶标。本提案中概述的工作将定义 PER2和CK1如何形成稳定的复合体并决定其调控的分子起源 复合体受温度的影响。这个项目将为我提供细胞培养和小型 分子抑制物表征，同时也建立在最初在研究生期间发展的核磁共振技能 学校让我为独立科学家的职业生涯做好更好的准备。CK1与PER2的相互作用 对于一个正常运行的时钟来说是至关重要的，因此识别这种监管机制的细节具有 为治疗由基因干扰的昼夜节律紊乱的人提供新治疗策略的可能性 失调、时差和倒班工作。

Project summary Circadian rhythms govern most behavioral and physiological processes to coincide with the 24-hour solar day. Disruption of circadian rhythms leads to increased risk for depression, cardiovascular disease, and cancer. The protein Period 2 (PER2) sits at the center of the mammalian molecular clock, and the levels of PER2 are tightly regulated through phosphorylation by casein kinase 1 delta (CK1). Mutation of a phosphorylation site destabilizes PER2, shortens circadian period, and leads to familial advanced sleep phase (FASP) syndrome in humans. Additionally, phosphorylation of PER2 here by CK1 may underlie temperature compensation, which allows the clock to generate ~24-hour timing independently of temperature. The molecular details of how CK1 interacts with PER2 to promote this stabilizing phosphorylation and how this interaction contributes to temperature compensation are poorly understood. The ​long-term objective​ of my research is to understand how CK1 phosphorylation of PER2 regulates the mammalian circadian clock. This proposal focuses on determining how CK1 interacts with PER2 to form a stable, stoichiometric complex and how it contributes to temperature compensation. My ​central​ ​hypothesis​ is that temperature compensation in the mammalian circadian clock is achieved via changes in dynamics in CK1 that influence its ability to form a stable complex with PER2 and/or phosphorylate the FASP region. The first aim of this project is to determine the molecular details of the PER2- CK1 interaction that contribute to complex formation. Using biophysical techniques, I will structurally characterize the complex and the binding of a new small molecule inhibitor that disrupts the PER2- CK1 interaction. The second aim of this project is to determine how altered protein dynamics contribute to temperature compensation in the clock. The dynamics may be occurring on several timescales, which makes it an appropriate target to study using solution NMR spectroscopy. This work outlined in this proposal will define how PER2 and CK1 form a stable complex and determine the molecular origins of how regulation of this complex is impacted by temperature. This project will provide me with training in cell culture and small molecule inhibitor characterization, while also building on skills in NMR initially developed during graduate school to better prepare me for a career as an independent scientist. The interaction between CK1 and PER2 is crucial to a properly functioning clock, so identifying the details of this regulatory mechanism has the potential to inform new therapeutic strategies to treat people with circadian rhythms disrupted by genetic disorders, jet lag, and shift work.