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可以测试键的合成振荡器或开关 内源性生物学途径的设计原理并纠正错误调节的途径。 哺乳动物的昼夜振荡器涉及许多机制，但它们的环状和 互连使测试哪些机制确实至关重要。建立合成 使用与内源性相似的设计原理在哺乳动物细胞中的昼夜节律时钟将是最大的 直接和令人信服的方法来测试和识别关键机制。这种创新的结构也将使我们 了解和开发针对昼夜节律障碍和其他类型的神经系统或 由时钟故障引起的生理功能障碍。基于数学建模和过去的实验， 构建合成的绝对需要每个蛋白质的限速时钟组件的独特参数 昼夜节律。这里的中心假设是许多异源转录反馈回路可以 如果反馈抑制是由蛋白质介导的，则产生自主昼夜节律 可以生成必要的昼夜节律参数，例如时间延迟和非线性。 AIM 1。在哺乳动物细胞中产生完全合成的昼夜节律。合成转录反馈 循环将使用酵母衍生的GAL4-UAS-GAL80系统生成：GAL4将激活转录 UAS-GAL80，但后来将被Gal80抑制以关闭反馈循环。每个将融合到gal80到 确保 - 如果我们的假设是正确的，那么这种反馈抑制是以昼夜节律介导的。它有 在几种情况下证明了Per的昼夜活动不受与其他融合的影响 蛋白质，例如荧光素酶和金星。合成振荡器的功能将由 像以前完成的那样，实时测量UAS-荧光素酶或GFP的节奏。根据 数学预测，一旦通过临时重置，该电路将产生强大的昼夜节律 从第二个可诱导的启动子引起的PER2-GAL80表达。 目标2。从对24小时振荡至关重要的PER2蛋白中确定一个新的基序。我们的初步 数据表明，PER2的昼夜节律取决于PER2的特定领域 受到独特的翻译后调节。 Degron已被设计为合成电路 在没有特定时间动力学的情况下提供增强的降解。我们认为，确定的“ 24小时域” 这项研究可以设计到其他蛋白质和电路中，以将昼夜节律授予电路， 无需编码每个蛋白质的全长。此外，这种主题和对其作用的机制 将是调节昼夜节律时钟的治疗的主要目标。