Regulation of mammalian cell physiology by a novel synthetic circadian clock

通过新型合成生物钟调节哺乳动物细胞生理学

• 批准号: 9226127
• 负责人: CHOOGON LEE
• 金额: $ 22.8万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226127
• 关键词: Affect; Amino Acids; Behavior Control; Binding; Biological; Biological Models; Bioluminescence; Biomedical Engineering; Biotechnology; Blindness; CSNK1A1 gene; Cell Culture Techniques; Cell Cycle; Cell physiology; Cells; Chimeric Proteins; Circadian Rhythms; Complementary DNA; DNA Sequence Alteration; Data; Disease; Drug Targeting; Engineering; Ensure; Feedback; Fluorescence; Functional disorder; Generations; Genes; Genetic; Genetic Transcription; Goals; Half-Life; Hour; Kinetics; Length; Luciferases; Mammalian Cell; Mammals; Measures; Mediating; Mus; Mutant Strains Mice; Nature; Neurologic; Nuclear; Pathway interactions; Periodicity; Pharmaceutical Preparations; Phase; Physiological; Physiology; Post-Translational Regulation; Property; Proteins; Regulation; Reporter; Research; Sleep; Sleep Disorders; Structure; System; Testing; Tetanus Helper Peptide; Therapeutic; Time; Transcriptional Regulation; Venus; Work; Yeasts; abstracting; base; circadian pacemaker; design; genetic approach; inhibitor/antagonist; innovation; insight; insulin secretion; mathematical model; nervous system disorder; novel; promoter; research study; reverse genetics; shift work; targeted treatment

Project Summary/Abstract Objectives and Rationale: Our long-term goal is to engineer synthetic oscillators or switches that can test key design principles of endogenous biological pathways and correct the pathways when they are mis-regulated. Many mechanisms are involved in the mammalian circadian oscillator, but their cyclic nature and interconnections make it very difficult to test which mechanisms are truly essential. Building a synthetic circadian clock in mammalian cells using similar design principles as the endogenous ones would be the most direct and convincing way to test and identify key mechanisms. This innovative construct would also enable us to understand and develop new treatments for circadian sleep disorders and other types of neurological or physiological dysfunction caused by faulty clocks. Based on mathematical modeling and past experiments, the unique parameters of the rate-limiting clock component PER protein are absolutely required to build a synthetic circadian clock. The central hypothesis here is that many heterologous transcriptional feedback loops can produce autonomous circadian rhythms if the feedback inhibition is mediated by PER protein because PER can generate necessary circadian parameters such as time delay and nonlinearity. Aim 1. Generate a fully synthetic circadian clock in mammalian cells. A synthetic transcriptional feedback loop will be generated using the yeast-derived GAL4-UAS-GAL80 system: GAL4 will activate transcription of UAS-Gal80 but later will be inhibited by GAL80 to close the feedback loop. PER will be fused to GAL80 to ensure—if our hypothesis is correct—that this feedback inhibition is mediated in a circadian manner. It has been demonstrated in several cases that the circadian activities of PER are not affected by fusion with other proteins such as Luciferase and Venus. The functionality of the synthetic oscillator will be assessed by measuring rhythms from UAS-Luciferase or GFP in real time, as it has been done previously. According to mathematical predictions, the circuit will produce robust circadian rhythms once it is reset by temporarily inducing Per2-Gal80 expression from a second, drug-inducible promoter. Aim 2. Identify a novel motif from the PER2 protein critical for the 24-hour oscillations. Our preliminary data suggest that the circadian rhythmicity of PER2 is dependent upon a specific domain of PER2 that is subjected to unique posttranslational regulation. Degrons have been engineered into synthetic circuits to provide enhanced degradation without specific time kinetics. We believe that the “24-hour domain” identified by this study could be engineered into other proteins and circuits to confer a circadian property to the circuits, without incorporating the full-length PER protein. Furthermore, this motif and the mechanisms that act upon it would be a prime target for therapeutics to modulate the circadian clock.

项目概要/摘要 目标和理由：我们的长期目标是设计可以测试关键的合成振荡器或开关 内源性生物通路的设计原则，并在通路被错误调节时进行纠正。 哺乳动物昼夜节律振荡器涉及许多机制，但它们的循环性质和 互连使得测试哪些机制真正重要变得非常困难。构建一个合成 哺乳动物细胞中的生物钟使用与内源性生物钟类似的设计原理将是最有效的 测试和识别关键机制的直接且令人信服的方法。这种创新的结构也将使我们能够 了解和开发针对昼夜节律睡眠障碍和其他类型的神经或疾病的新疗法 时钟故障导致的生理功能障碍。基于数学模型和过去的实验， 速率限制时钟组件 PER 蛋白的独特参数对于构建合成的 生物钟。这里的中心假设是许多异源转录反馈环可以 如果反馈抑制是由 PER 蛋白介导的，则产生自主的昼夜节律，因为 PER 可以生成必要的昼夜节律参数，例如时间延迟和非线性。 目标 1. 在哺乳动物细胞中生成完全合成的生物钟。合成转录反馈 环将使用酵母衍生的 GAL4-UAS-GAL80 系统生成：GAL4 将激活 UAS-Gal80 但稍后会被 GAL80 抑制以关闭反馈回路。 PER 将与 GAL80 融合 确保——如果我们的假设是正确的——这种反馈抑制是以昼夜节律方式介导的。它有 多个案例已证明 PER 的昼夜节律活动不受与其他物质融合的影响 蛋白质，例如荧光素酶和维纳斯。合成振荡器的功能将通过以下方式评估 正如之前所做的那样，实时测量 UAS-荧光素酶或 GFP 的节律。根据 根据数学预测，一旦暂时重置，电路将产生强大的昼夜节律 从第二个药物诱导型启动子诱导 Per2-Gal80 表达。 目标 2. 从 PER2 蛋白中识别出对 24 小时振荡至关重要的新基序。我们的初步 数据表明 PER2 的昼夜节律取决于 PER2 的特定域，即 受到独特的翻译后调控。降解分子已被设计到合成电路中 无需特定时间动力学即可增强降解。我们认为，所确定的“24小时域” 这项研究可以被工程化到其他蛋白质和电路中，从而赋予电路昼夜节律特性， 不掺入全长 PER 蛋白。此外，这个主题和作用于它的机制 将是调节生物钟治疗的主要目标。