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: 9015773
• 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/9015773
• 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; Health; 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; rate of change; 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）：调节蛋白质周转对于所有真核生物的细胞决策至关重要。降解速率通常充当协调细胞内和细胞间反应的起搏器，并且在人类疾病中经常失调。然而，我们知道的显着很少是什么控制底物降解动力学。一个可能的原因是缺乏可用的模型，高分辨率的结构功能分析的周转率。SCF类E3泛素连接酶在动物、植物和真菌中高度保守。在这些复合物中，F-盒作为底物识别亚基将泛素添加到不同的靶点。我们建议使用SCF参与生长素的反应，在植物生物学的几乎每个方面的心脏，作为一个模型，调查E3功能的一般原则。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，并促进了我们在酵母中生长素诱导的降解工程。使用该系统，我们发现F盒极大地影响降解速率，并且先前未表征的序列以底物特异性方式加速或减速降解。我们的中心假设是，广泛的生长素诱导的底物降解动力学是实现广泛的生长素调节的细胞反应的关键，并且这些动力学是由不同的结构域内的F-盒和基板编码。为了验证这一假设，我们提出：（1）确定F盒和底物中降解速率的决定因素。我们已经确定了两个新的底物域控制降解速率和几个新的F-box域的兴趣。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，以测试该参数决定降解速率的程度。(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。我们现在可以量化和数学模型底物周转和下游响应之间的关系。这项技术使我们能够研究以前难以解决的问题，如底物二聚化对生长素反应的影响。(3)量化不同退化率对发展的影响。我们从转基因植物中获得的初步数据将底物降解速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，以分析发育进程如何受到底物周转时间的影响。总之，拟议的工作将为E3在生长素反应中的功能提供一个机制框架，促进识别影响的网络架构 信号的灵敏度和持续时间，并可能提供洞察E3的基本属性：底物相互作用。这些见解可以为我们了解与人类疾病相关的E3提供信息，并指导未来使用生长素成分设计用于诊断或治疗应用的合成电路。