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: 9341405
• 负责人: CHOOGON LEE
• 金额: $ 19万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9341405
• 关键词: 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; Mathematics; 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; base; circadian pacemaker; design; experimental study; genetic approach; inhibitor/antagonist; innovation; insight; insulin secretion; mathematical model; nervous system disorder; novel; promoter; 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.

项目摘要/摘要 目标和基本原理：我们的长期目标是设计能够测试按键的合成振荡器或开关 内源性生物通路的设计原则，并在错误调节时纠正这些通路。 哺乳动物的昼夜节律振荡器涉及许多机制，但它们的周期性和 互联互通使得测试哪些机制真正至关重要变得非常困难。建造一个合成的 哺乳动物细胞中的生物钟使用与内源性时钟相似的设计原理将是最 以直接和令人信服的方式测试和确定关键机制。这种创新的构造也将使我们能够 了解并开发新的治疗昼夜睡眠障碍和其他类型的神经或精神疾病的方法 由于时钟故障而导致的生理功能障碍。基于数学建模和过去的实验， 每种蛋白质的限速时钟分量的唯一参数是绝对需要的，以建立一个合成的 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 可以产生必要的昼夜节律参数，如时延和非线性。 目的1.在哺乳动物细胞中产生一个完全合成的生物钟。一种合成转录反馈 将使用酵母衍生的GAL4-UAS-GAL80系统产生环：GAL4将激活转录 UAS-Gal80但之后将被GAL80抑制以闭合反馈回路。PER将与GAL80融合，以 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的方式调节的。它有 已经在几个案例中证明了PER的昼夜活动不受与其他 如荧光素酶和金星等蛋白质。合成振荡器的功能将通过以下方式进行评估 实时测量UAS-荧光素酶或GFP的节律，就像以前做的那样。根据 数学预测，一旦电路被临时重置，它将产生强大的昼夜节律 从第二个药物诱导启动子诱导PER2-Gal80的表达。 目的2.从PER2蛋白中鉴定出一个对24小时振荡至关重要的新基序。我们的预赛 数据表明，PER2的昼夜节律性依赖于PER2的一个特定结构域，即 受到独特的翻译后监管。Deegron已被设计成合成电路以 提供强化的降解，而不需要特定的时间动力学。我们认为，由 这项研究可以被工程到其他蛋白质和电路中，以赋予电路昼夜节律特性， 而不包含每种蛋白质的全长。此外，这一主题和作用于它的机制 将成为治疗学家调节生物钟的首要目标。