Discovering protein degradation mechanisms that regulate the plant circadian clock

项目摘要/摘要 生物钟是使生物过程与环境同步所必需的。一个恰到好处的时机 时钟依赖于时钟蛋白的快速产生和破坏。尽管如此，很少有蛋白质降解 已经发现了调节植物生物钟的机制。这很可能是由于基因 控制蛋白质泛素化的E3泛素连接酶之间的冗余。拟南芥一直是一个 利用正演实时成像发现昼夜节律钟分子成分的强大系统 和反向基因筛查。我的实验室利用这些优势进行了两次反向遗传 E3泛素连接酶家族的筛选，克服了传统的遗传冗余和 确定一系列新的生物钟功能调节器。这份建议书描述了设计和 该屏幕的执行以及新发现的时钟调节器的早期功能表征。 功能鉴定依赖于我们简化的工作流程，使我们能够快速确定E3 泛素连接酶底物蛋白，验证这些潜在的底物，并进行遗传和生化 实验证明了它们在时钟功能中的作用。然后，提案描述了我们的两个主要实验室 下一步目标：1)完成拟议的屏幕和2)新的 发现了时钟调节器。这些研究将确定蛋白质降解机制如何有助于 时钟感应外部信号，保持24小时节律，并连接到下游有节奏的生物 流程。 生物钟调节基本的生物过程，包括光合作用、新陈代谢、 防御和增长。因此，我们开展的工作将产生深远的影响，因为：1)它将提供 在植物中产生强大的24小时时钟所必需的基本分子构件，2)它将 作为非植物系统中类似研究的框架，以及3)它将提供更全面的 理解覆盖时钟转录反馈环的翻译后机制。 这项提议的成功完成将揭示翻译后退化机制如何调查 细胞环境控制生物钟转录网络的变化并提供精确的 时钟控制的生物过程的计时。从长远来看，这项工作将增加我们对 对植物关键生物过程的调控，但它也将转化为对时钟的更好理解 人类的功能和时钟相关疾病。

Project Summary/Abstract The circadian clock is necessary to synchronize biological processes with the environment. A properly timed clock relies on rapid creation and destruction of clock proteins. Despite this, few protein degradation mechanisms have been discovered that regulate the circadian clock of plants. This is likely due to genetic redundancy amongst the E3 ubiquitin ligases that control protein ubiquitylation. Arabidopsis has served as a powerful system for discovering molecular components of the circadian clock using live imaging for forward and reverse genetic screens. My laboratory has leveraged these advantages to perform two reverse genetic screens of E3 ubiquitin ligase families, overcoming traditional problems with genetic redundancy and identifying a host of new regulators of circadian clock function. This proposal describes the design and execution of this screen and the early functional characterization of newly discovered clock regulators. Functional characterization relies on our streamlined workflow that allows us to rapidly determine the E3 ubiquitin ligase substrate proteins, validate these potential substrates, and perform genetic and biochemical experiments demonstrating their role in clock function. The proposal then describes our two main laboratory goals moving forward: 1) completion of the proposed screens and 2) functional characterization of the newly discovered clock regulators. These studies will determine how protein degradation mechanisms can help clocks sense external signals, maintain a 24 hour rhythm, and connect to downstream rhythmic biological processes. The circadian clock regulates fundamental biological processes including photosynthesis, metabolism, defense, and growth. Thus, the work that we perform will have far-reaching impacts because: 1) it will provide the basic molecular building blocks that are necessary to generate a robust 24 hour clock in plants, 2) it will serve as a framework for similar studies in non-plant systems, and 3) it will provide a more comprehensive understanding of the post-translational mechanisms that overlay transcriptional feedback loops of clocks. Successful completion of this proposal will reveal how post-translational degradation mechanisms can survey cellular environments to control changes in transcriptional networks of circadian clocks and provide precise timing to clock-controlled biological processes. In the longer term, this work will increase our understanding of the regulation of critical biological processes in plants, but it will also translate to better understanding of clock function and clock-related diseases in humans.