Regulation of mammalian cell physiology by a novel synthetic circadian clock

项目总结/摘要 目标和基本原理：我们的长期目标是设计合成振荡器或开关，可以测试关键 设计内源性生物学途径的原则，并在这些途径被错误调节时进行纠正。 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 相互联系使得很难检验哪些机制是真正必要的。建造一个合成人 在哺乳动物细胞中使用与内源性生物钟相似的设计原理， 直接和令人信服的方式来测试和识别关键机制。这一创新的结构也将使我们能够 了解和开发新的治疗昼夜睡眠障碍和其他类型的神经或 由时钟故障引起的生理机能障碍。基于数学建模和过去的实验， 限速时钟组件PER蛋白的独特参数是绝对需要建立一个合成的 生物钟这里的中心假设是，许多异源转录反馈环可以 产生自主的昼夜节律，如果反馈抑制是由PER蛋白介导的， 可以产生必要的昼夜节律参数，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。一种合成的转录反馈 将使用酵母衍生的GAL 4-UAS-GAL 80系统生成环：GAL 4将激活 UAS-Gal 80，但稍后将被GAL 80抑制以关闭反馈回路。PER将与GAL 80融合， 确保-如果我们的假设是正确的-这种反馈抑制介导的昼夜节律的方式。它有 在一些情况下已经证明，PER的昼夜活动不受与其他生物的融合的影响。 蛋白质如荧光素酶和金星。合成振荡器的功能将通过以下方式进行评估： 如以前所做的那样，在真实的时间内测量来自UAS-荧光素酶或GFP的节律。根据 数学预测，一旦电路被暂时重置，它将产生强大的昼夜节律。 从第二药物诱导型启动子诱导Per 2-Gal 80表达。 目标二。从PER 2蛋白中识别出一个对24小时振荡至关重要的新基序。我们的初步 数据表明，PER 2的昼夜节律性依赖于PER 2的一个特定结构域， 受到独特的翻译后调节。降解决定子已经被设计到合成电路中， 提供增强的降解而没有特定的时间动力学。我们认为， 这项研究可以被设计到其他蛋白质和电路中以赋予电路昼夜节律特性， 而不掺入全长PER蛋白。此外，这个主题和作用于它的机制 将是治疗调节生物钟的主要目标。

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.