Control of protein degradation and transcriptional dynamics in the auxin response

蛋白质周转率可以作为一个起搏器，协调细胞内和细胞间的反应， 在人类疾病中经常失调。然而，我们对控制底物的物质知之甚少， 降解动力学或这些动力学如何转化为下游响应。一个可能的原因是 缺乏降解和转录的高分辨率结构-功能分析的可用模型 activation. SCF类E3泛素连接酶在动物、植物和真菌中高度保守。我们 我建议使用一种参与生长素反应的SCF，它几乎是植物生物学各个方面的核心， 模型研究E3功能的一般原则，并将该功能与转录 激活和形态发生。生长素途径中的小分子引发的降解提供了独特的 这些研究的优势，并促进了我们的工程生长素诱导的降解， 酵母中的转录激活。对这个系统的研究导致了我们的中心假设：生长素系统 在植物中作为一个通用的发育计时器，类似的逻辑电路可能在大多数真核生物中起作用。 为了验证这一假设，我们建议：（1）定义降解变化的决定因素和相关性 rates.我们已经确定了E3和底物组分的几个感兴趣的领域，并正在使用 将单个残留物与降解动态联系起来的合成和计算工具。(2)量化 降解速率对转录抑制的影响。我们已经扩展了我们的合成分析， 生长素诱导的转录。我们现在可以定量地追踪底物周转 和下游响应。这项技术使我们能够研究以前难以解决的问题 比如去除辅阻遏物是如何与转录激活相结合的。(3)偶联细胞 降解定时器到发育转变。我们已经在转基因植物中发现， 降解速度决定了侧器官发育的速度。我们将使用多种互补的方法 分析这些植物的转录组，以阐明底物周转的时间如何调节 以细胞类型依赖的方式发育进展。总之，拟议的工作将提供一个 E3在生长素反应中的作用机制框架，并可能提供对基本原理的见解 E3的性质：底物相互作用和下游事件。这些见解可以帮助我们理解 与人类疾病相关的E3，以及指导未来使用生长素合成电路的设计 用于治疗应用的组分。

The rate of protein turnover can act as a pacemaker to coordinate responses within and between cells, and is frequently dysregulated in human disease. Yet we know remarkably little about what controls substrate degradation kinetics or how these kinetics translate into downstream responses. One possible reason is the lack of available models for high- resolution structure-function analysis of degradation and transcriptional activation. The SCF class of E3 ubiquitin ligases is highly conserved among animals, plants and fungi. We propose to use an SCF involved in auxin response, at the heart of nearly every aspect of plant biology, as a model to investigate general principles underlying E3 function and connect that function to transcriptional activation and morphogenesis. The small-molecule triggered degradation in the auxin pathway offers a unique advantage for these studies, and has facilitated our engineering of auxin-induced degradation and transcriptional activation in yeast. Work with this system has led to our central hypothesis: the auxin system functions as a universal developmental timer in plants, and similar logic circuits likely act in most eukaryotes. To test this hypothesis, we propose to: (1) Define the determinants and relevance of variation in degradation rates. We have already identified several domains of interest in E3 and substrate components, and are using synthetic and computational tools to connect individual residues to degradation dynamics. (2) Quantify the impact of degradation rate on transcriptional repression. We have extended our synthetic assays to include auxin-induced transcription. We can now quantitatively track the molecular events between substrate turnover and downstream responses over time. This technology enables our study of previously intractable problems like how the removal of co-repressors is integrated with transcriptional activation. (3) Couple cellular degradation timers to developmental transitions. We have shown in transgenic plants that substrate degradation rate sets the pace of lateral organ development. We will use multiple, complementary approaches to analyze the transcriptome of these plants to elucidate how the timing of substrate turnover regulates developmental progression in a cell-type-dependent manner. Together, the proposed work will provide a mechanistic framework for E3 function in the auxin response and potentially provide insights into fundamental properties of E3:substrate interactions and downstream events. These insights can inform our understanding of E3s associated with human disease, as well as guiding future design of synthetic circuits using auxin components for therapeutic applications.