Structural Study of GTPase Regulators and Effectors

描述(由申请人提供)： 从WASP/WAVE蛋白到Arp2/3复合体的保守通路在广泛的细胞过程中控制着肌动蛋白的动态。在上一个周期中，我们了解了WASP是如何被小分子wikostatin抑制的，并整合了来自CDC42 GTP酶和激酶/磷酸酶的信号，我们建立了WASP自抑制的定量热力学模型。在这里，我们将关注一种新的重要的调节机制，即WASP/WAVE蛋白通过寡聚作用使其对Arp2/3复合体的效力提高约100倍。这种戏剧性的影响影响了广泛的WASP/WAVE信号，我们将在本提案中对其进行广泛的探讨。在感染过程中，黄蜂被人类病原体和潜在的生物恐怖主义病原体--肠出血性大肠杆菌的EspFu蛋白激活。我们将确定WASP-EspFu复合体的结构，并使用生化和生物物理方法确定EspFu的多个序列重复之间的协同作用是否源于WASP齐聚。我们还将使用多学科方法来确定寡聚的相对重要性和在WASP激活过程中通过一组含有SH3的蛋白质解除自身抑制。我们已经重组了400 kDa的异五聚体波调节复合体(WRC)和来自人和苍蝇的各种亚复合体，并将使用这些强大的试剂来了解波是如何受到上游输入的控制，包括RAC和寡聚剂。这些研究将通过确定WRC的一个关键的三聚亚复合体的结构来补充。整个计划将揭示不同的正常和基于疾病的信号输入控制WASP和WAVE活动的机制。在整个过程中，我们将发展一个新的概念，即WASP/WAVE的活性是分级控制的；内层调节非活性状态和活性状态之间的平衡，外层调节活性状态与Arp2/3复合体的亲和力。这一概念将在一个共同的机制框架下统一该领域的大量工作，定量解释不同的信号如何汇聚在Arp2/3复合体上以产生复杂的肌动蛋白动力学。这项工作还将调和两种截然相反的WAVE监管模式，并揭示WASP/WAVE家族的共同和独特的监管原则。我们的发现将解决生物物理学、信号转导和细胞生物学中的基本问题，并可能为检测和治疗癌症、人类遗传疾病和细菌感染提供新的和改进的方法。我们的研究集中在了解连接Rho GTP酶和WASP/WAVE蛋白到Arp2/3复合体的信号通路。这些通路在许多正常的生物学过程和许多疾病中起着关键作用，包括转移性癌症、免疫紊乱和细菌/病毒感染。对这些蛋白质如何在这些途径中进行交流以及如何被细菌转移的了解，可能会导致许多疾病的诊断和治疗的新试剂。

DESCRIPTION (provided by applicant): Conserved pathways from WASP/WAVE proteins to the Arp2/3 complex control actin dynamics in a huge range of cellular processes. In the previous cycle we learned how WASP is inhibited by the small molecule wiskostatin and integrates signals from the Cdc42 GTPase and kinases/phosphatases, and we developed a quantitative thermodynamic model for WASP autoinhibition. Here, we will focus on a new and important regulatory mechanism, in which the potency of WASP/WAVE proteins toward Arp2/3 complex is increased ~100-fold through oligomerization. This dramatic effect impacts on a wide range of WASP/WAVE signals, and we will explore it broadly in this proposal. WASP is activated by the EspFu protein from the human pathogen and potential bioterrorism agent, Enterohemorrhagic E. coli, during infection. We will determine the structure of a WASP-EspFu complex and use biochemical and biophysical methods to determine whether cooperativity between the multiple sequence repeats of EspFu results from WASP oligomerization. We will also use a multi-disciplinary approach to determine the relative importance of oligomerization and relief of autoinhibition during WASP activation by a battery of SH3-containing proteins. We have reconstituted the 400 kDa hetero-pentameric WAVE Regulatory Complex (WRC) and various subcomplexes from humans and flies, and will use these powerful reagents to learn how WAVE is controlled by upstream inputs including Rac and oligomerizing agents. These investigations will be complemented by structure determination of a key trimeric subcomplex of the WRC. The overall program will reveal the mechanisms by which diverse normal and disease-based signaling inputs control the activity of WASP and WAVE. Throughout, we will develop a novel concept that WASP/WAVE activity is controlled hierarchically; an inner layer of regulation governs the equilibrium between inactive and active states, and an outer layer governs the affinity of the active state for Arp2/3 complex. This concept will unify a large body of work in the field under a common mechanistic framework, explaining quantitatively how disparate signals converge on Arp2/3 complex to generate complex actin dynamics. The work will also reconcile two opposing models of WAVE regulation, and reveal common and distinct regulatory principles across the WASP/WAVE family. Our findings will address fundamental questions in biophysics, signal transduction and cell biology and could suggest new and improved methods for the detection and treatment of cancer, human genetic disorders and bacterial infection. Our research focuses on understanding the signaling pathways that connect Rho GTPases to WASP/WAVE proteins to the Arp2/3 complex. These pathways are critically involved in many normal biological processes and in numerous diseases, including metastatic cancer, immune disorders and bacterial/viral infection. An understanding of how the proteins communicate in these pathways, and can be diverted by bacteria, could lead to new agents for the diagnosis and treatment of many diseases.