Mechanisms of regulation of Arp2/3 complex

描述(申请人提供)：这项工作的长期目标是了解Arp2/3复合体调节的分子基础。Arp2/3复合体是肌动蛋白组装机械的重要组成部分，因为它能够响应细胞信号而使分枝的肌动蛋白细丝成核。Arp2/3复合体不仅参与了健康细胞的内吞和生长锥体运动等过程，还参与了细菌感染宿主细胞和肿瘤细胞转移的过程。Arp2/3复合体的活性在体内受到严格的调控，已经发现了许多该复合体的激活剂和抑制剂，包括黄蜂/疤痕家族蛋白和冠状病毒家族蛋白。尽管Arp2/3复合体具有核心重要性，但由于缺乏该复合体部分或全部激活状态的生化和高分辨结构信息，其激活的分子机制仍不清楚。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复合体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实现这一目标：1.确定允许Arp2/3复合体与肌动蛋白的母丝和子丝结合的关键结构元件；确定黄蜂/疤痕家族蛋白如何激活Arp2/3复合体；确定柯罗宁蛋白如何抑制Arp2/3复合体。我们的方法将是使用突变分析和生化分析来精确地定位S.pombe Arp2/3复合体与肌动蛋白母丝和子丝的相互作用。我们将使用质谱学、X射线结晶学和突变分析来确定黄蜂/SCAR家族蛋白与Arp2/3复合体结合的位置，以及它们如何为子丝招募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冠状蛋白如何抑制Arp2/3复合体，并影响其与肌动蛋白细丝的相互作用。 公共卫生相关性：在这项工作中，我们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感染人类细胞，癌细胞依靠这种机制传播。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理解人类的疾病状态以及如何治疗它们。

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.