Control of protein degradation dynamics in the auxin response

生长素反应中蛋白质降解动力学的控制

• 批准号: 8695018
• 负责人: JENNIFER L NEMHAUSER
• 金额: $ 30.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695018
• 关键词: Affect; Affinity; Animals; Antineoplastic Agents; Area; Auxins; Biochemical; Biological Assay; Biology; Cells; Complex; Crystallography; Cullin Proteins; Data; Decision Making; Development; Diabetes Mellitus; Diagnosis; Diagnostic; Dimerization; Drug Targeting; Engineering; Eukaryota; Event; Evolution; F Box Domain; F-Box Proteins; Fluorescence; Future; Genetic Transcription; Goals; Heart; Heterodimerization; Homeostasis; Homodimerization; Human; Human Development; Human Genome; Kinetics; Link; Logic; Maintenance; Malignant Neoplasms; Maps; Measures; Mediating; Modeling; Molecular; Nature; Neurodegenerative Disorders; Organ; Output; Pacemakers; Pathway interactions; Phosphorylation; Plants; Process; Property; Proteasome Inhibitor; Protein Kinase; Proteins; Resolution; Series; Signal Transduction; Specificity; Speed; Structure; Substrate Domain; Substrate Interaction; System; Technology; Testing; Therapeutic; Time; Transgenic Organisms; Transgenic Plants; Ubiquitin; Variant; Work; Yeasts; design; drug discovery; fungus; human disease; insight; interest; network architecture; novel; plant fungi; programs; protein degradation; public health relevance; receptor; research study; response; small molecule; stem cell fate specification; therapy design; tool; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Regulated protein turnover is critical for cellular decision-making in all eukaryotes. The rate of degradation often acts 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. One possible reason is the lack of available models for high resolution structure-function analysis of turnover rates. The SCF class of E3 ubiquitin ligases is highly conserved among animals, plants and fungi. Within these complexes, F-boxes act as substrate-recognition subunits to add ubiquitin to diverse targets. 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. 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 in yeast. Using this system, we discovered that F-boxes greatly impact the rate of degradation and that previously uncharacterized sequences accelerated or decelerated degradation in a substrate-specific manner. Our central hypothesis is that the wide range of auxin-induced substrate degradation dynamics are critical for achieving the wide range of auxin-regulated cellular responses, and that these dynamics are encoded by distinct domains within F-boxes and substrates. To test this hypothesis, we propose to: (1) identify determinants of degradation rate in F-boxes and substrates. We have already identified two new substrate domains controlling degradation rates and several new F-box domains of interest. Structural studies are underway to investigate the functional relevance of these domains, and we are using a number of tools to quantify complex affinity to test the degree to which this parameter dictates degradation rates. (2) quantify the impact of varying degradation rate on transcription. We have extended our synthetic assays to include auxin- induced transcription. We can now quantify and mathematically model the relationship between substrate turnover and downstream responses. This technology enables our study of previously intractable problems like the impact of substrate dimerization on auxin responses. (3) quantify the impact of varying degradation rate on development. We have preliminary data from transgenic plants linking substrate degradation rate to the proper development of new organs. We will perform in-depth cellular and molecular phenotypic analysis of these plants to analyze how developmental progression is affected by timing of substrate turnover. Together, the proposed work will provide a mechanistic framework for E3 function in the auxin response, facilitate identification of network architectures impacting signal sensitivity and duration, and potentially provide insights into fundamental properties of E3: substrate interactions. 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 diagnostic or therapeutic applications.

描述(由申请人提供)：在所有真核生物中，受调控的蛋白质周转对细胞决策至关重要。降解率经常起到协调细胞内和细胞间反应的起搏器的作用，在人类疾病中经常处于失调状态。然而，我们对控制底物降解动力学的因素知之甚少。一个可能的原因是缺乏可用于高分辨率结构-功能分析的流动率模型。SCF类E3泛素连接酶在动物、植物和真菌中高度保守。在这些复合体中，F-box充当底物识别亚单位，将泛素添加到不同的靶标上。我们建议使用一个参与生长素反应的SCF作为一个模型来研究E3功能的一般原理，生长素反应几乎是植物生物学各个方面的核心。生长素途径中的小分子引发的降解为这些研究提供了独特的优势，并促进了我们对生长素诱导酵母降解的工程设计。使用这个系统，我们发现F-box对降解速度有很大的影响，以前没有描述的序列以底物特有的方式加速或减速降解。我们的中心假设是，生长素诱导的底物降解动力学是实现生长素调节的广泛细胞反应的关键，这些动力学是由F盒和底物中不同的结构域编码的。为了验证这一假设，我们建议：(1)确定F盒和底物中降解率的决定因素。我们已经确定了两个控制降解速率的新底物结构域和几个新的感兴趣的F-box结构域。结构研究正在进行中，以调查这些结构域的功能相关性，我们正在使用一些工具来量化复杂的亲和力，以测试这个参数决定降解率的程度。(2)量化不同降解速率对转录的影响。我们已经扩展了我们的合成分析，包括生长素诱导的转录。我们现在可以对底物周转和下游反应之间的关系进行量化和数学建模。这项技术使我们能够研究以前难以解决的问题，如底物二聚对生长素反应的影响。(3)量化不同退化速度对发展的影响。我们有来自转基因植物的初步数据，将底物降解率与新器官的正常发育联系起来。我们将对这些植物进行深入的细胞和分子表型分析，以分析底物周转时间对发育进程的影响。总之，拟议的工作将为E3在生长素响应中的作用提供一个机械框架，促进识别影响网络体系结构 信号灵敏度和持续时间，并潜在地提供对E3的基本属性的洞察：底物相互作用。这些见解可以帮助我们理解与人类疾病相关的E3，并指导未来使用生长素元件设计用于诊断或治疗应用的合成电路。