Discovering protein degradation mechanisms that regulate the plant circadian clock

项目总结/摘要 生物钟是使生物过程与环境同步所必需的。一个适当的时间 时钟依赖于时钟蛋白的快速产生和破坏。尽管如此， 已经发现了调节植物生物钟的机制。这可能是由于遗传 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 一个强大的系统，用于发现生物钟的分子组成部分，使用实时成像， 和反向基因筛选我的实验室利用这些优势， 筛选E3泛素连接酶家族，克服了遗传冗余的传统问题， 确定了一系列新的生物钟功能调节器。本提案描述了设计和 该屏幕的执行和新发现的时钟调节器的早期功能特性。 功能表征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，并进行遗传和生物化学 实验证明它们在时钟功能中的作用。该提案然后描述了我们的两个主要实验室 今后的目标：1）完成拟议的筛选和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帮助 生物钟感知外部信号，保持24小时节律，并连接到下游节律性生物信号。 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，我们所做的工作将产生深远的影响，因为：1）它将提供 基本的分子构建模块，这是在植物中产生一个强大的24小时生物钟所必需的，2）它将 作为非植物系统中类似研究的框架，3）它将提供更全面的 理解翻译后机制，覆盖转录反馈循环的时钟。 成功完成这项提案将揭示如何翻译后降解机制可以调查 细胞环境来控制生物钟转录网络的变化，并提供精确的 生物钟控制的生物过程。从长远来看，这项工作将增加我们对 调节植物中的关键生物过程，但它也将转化为更好地理解时钟 与生物钟有关的疾病。

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.