Mechanisms of regulation of Arp2/3 complex

Arp2/3 复合体的调控机制

基本信息

批准号：
8291013
负责人：
Bradley J Nolen
金额：
$ 26.3万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8291013
关键词：
Actins Affect Bacteria Bacterial Infections Binding Biochemical Biological Biological Assay Cancerous Cell Nucleus Cell physiology Cells Cellular Structures Cicatrix Complex Coupling Crystallography Data Daughter Defect Elements Endocytosis Face Family Filament Fission Yeast Foundations Goals Growth Growth Cones Human In Vitro Indium Kinetics Maps Mass Spectrum Analysis Mediating Membrane Methods Microfilaments Minus End of the Actin Filament Modeling Molecular Molecular Conformation Molecular Models Mothers Movement Mutation Neoplasm Metastasis Organelles Play Process Protein Binding Protein Family Reaction Recruitment Activity Regulation Resolution Role Side Signal Transduction Structure Techniques Testing Virus Work Yeasts base cell motility coronin protein genetic regulatory protein improved in vivo inhibitor/antagonist molecular modeling monomer pathogen polymerization public health relevance response

项目摘要

DESCRIPTION (provided by applicant): The long term goal of this work is to understand the molecular basis for the regulation of Arp2/3 complex. Arp2/3 complex is an essential component of the actin assembly machinery because of its ability to nucleate branched actin filaments in response to cellular signals. Arp2/3 complex is involved in a number of processes that occur in healthy cells, such as endocytosis and motility of growth cones, but also plays roles in host cell infection by bacterial pathogens and the metastasis of tumor cells. The activity of Arp2/3 complex is tightly regulated in vivo, and numerous activators and inhibitors of the complex have been discovered, including WASp/Scar family proteins and coronin family proteins. Despite the central importance of the Arp2/3 complex, the molecular mechanism of its activation is still unknown due to the lack of biochemical and high resolution structural information of partially or fully activated states of the complex. Here we propose to use structural, biochemical and cell biological approaches to determine a precise molecular mechanism by which Arp2/3 complex and its regulatory proteins create branched actin networks. We will pursue this goal through the following three specific aims: 1.) Determine the key structural elements that allow Arp2/3 complex to bind to mother and daughter filaments of actin; 2.) Determine how WASp/Scar family proteins activate Arp2/3 complex; and 3.) Determine how Coronin proteins inhibit Arp2/3 complex. Our approach will be to use mutational analysis and biochemical assays to precisely map the interactions of S. pombe Arp2/3 complex with mother and daughter filaments of actin. We will use mass spectrometry, x-ray crystallography and mutational analysis to determine where WASp/Scar family proteins bind to Arp2/3 complex and how they recruit the first actin monomer for the daughter filament. Finally, we will use biochemical and structural methods to determine how yeast coronin proteins inhibit Arp2/3 complex and influence its interactions with actin filaments. PUBLIC HEALTH RELEVANCE: In this work, we are studying at the molecular level cellular machinery that controls actin polymerization. Bacteria and viruses use this machinery to infect human cells and cancerous cells depend on it to spread. Therefore, improving our understanding of the molecules that constitute this machinery will contribute to our understanding of diseased states in humans and how to treat them.

描述（由申请人提供）：这项工作的长期目标是了解 Arp2/3 复合物调节的分子基础。 Arp2/3 复合体是肌动蛋白组装机器的重要组成部分，因为它能够响应细胞信号使分支肌动蛋白丝成核。 Arp2/3复合物参与健康细胞中发生的许多过程，例如内吞作用和生长锥的运动，而且还在细菌病原体的宿主细胞感染和肿瘤细胞的转移中发挥作用。 Arp2/3 复合物的活性在体内受到严格调控，并且已发现该复合物的多种激活剂和抑制剂，包括 WASp/Scar 家族蛋白和 Coronin 家族蛋白。尽管 Arp2/3 复合物具有核心重要性，但由于缺乏复合物部分或完全激活状态的生化和高分辨率结构信息，其激活的分子机制仍然未知。在这里，我们建议使用结构、生化和细胞生物学方法来确定 Arp2/3 复合物及其调节蛋白创建分支肌动蛋白网络的精确分子机制。我们将通过以下三个具体目标来实现这一目标：1.）确定Arp2/3复合物与肌动蛋白母丝和子丝结合的关键结构元件； 2.) 确定WASp/Scar家族蛋白如何激活Arp2/3复合物； 3.) 确定 Coronin 蛋白如何抑制 Arp2/3 复合物。我们的方法是使用突变分析和生化测定来精确绘制粟酒裂殖酵母 Arp2/3 复合物与肌动蛋白母丝和子丝的相互作用。我们将使用质谱、X 射线晶体学和突变分析来确定 WASp/Scar 家族蛋白与 Arp2/3 复合物结合的位置以及它们如何为子丝募集第一个肌动蛋白单体。最后，我们将使用生化和结构方法来确定酵母 Coronin 蛋白如何抑制 Arp2/3 复合物并影响其与肌动蛋白丝的相互作用。公共卫生相关性：在这项工作中，我们正在分子水平上研究控制肌动蛋白聚合的细胞机制。细菌和病毒利用这种机制来感染人体细胞，癌细胞则依靠它来传播。因此，提高我们对构成这一机制的分子的理解将有助于我们了解人类的疾病状态以及如何治疗它们。