Structural Study of GTPase Regulators and Effectors

GTPase 调节器和效应器的结构研究

• 批准号: 7371663
• 负责人: Michael K Rosen
• 金额: $ 30.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371663
• 关键词: Actins; Adaptor Signaling Protein; Address; Affinity; Award; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; Biological Process; Biophysics; Bioterrorism; Cell Shape; Cell physiology; Cells; Cellular biology; Class; Complement; Complex; Data; Detection; Diagnosis; Dimerization; Disease; Disseminated Malignant Neoplasm; Elements; Equilibrium; Escherichia coli EHEC; Family; Filament; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Human; Hydrogen; Immune System Diseases; Infection; Investigation; Knowledge; Lead; Learning; Ligands; Lipids; Mediating; Methods; Microfilaments; Modeling; N-terminal; New Agents; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Proline-Rich Domain; Protein Binding; Protein Family; Protein Tyrosine Kinase; Proteins; Range; Reagent; Regulation; Relative (related person); Research; SH3 Domains; Signal Pathway; Signal Transduction; Signaling Molecule; Structure; Subgroup; Surface; Testing; Thermodynamics; Virus Diseases; WAVE protein; Wiskott-Aldrich Syndrome; Work; X-Ray Crystallography; base; cancer therapy; cell motility; concept; fly; foodborne; improved; inhibitor/antagonist; insight; member; milligram; novel; pathogen; pathogenic Escherichia coli; programs; protein activation; protein function; reconstitution; rho GTP-Binding Proteins; small molecule; tool; wiskostatin

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.

描述（由申请人提供）： 从WASP/WAVE蛋白到Arp 2/3复合物的保守途径控制着大量细胞过程中的肌动蛋白动力学。在前一个周期中，我们了解了WASP如何被小分子wiskostatin抑制，并整合了来自Cdc 42 GT3和激酶/磷酸酶的信号，我们开发了WASP自抑制的定量热力学模型。在这里，我们将集中在一个新的和重要的调节机制，其中WASP/WAVE蛋白对Arp 2/3复合物的效力通过寡聚化增加约100倍。这种戏剧性的效果影响了广泛的WASP/WAVE信号，我们将在本提案中广泛探讨。WASP被来自人类病原体和潜在生物恐怖剂肠出血性大肠杆菌的EspFu蛋白激活。大肠杆菌，在感染期间。我们将确定WASP-EspFu复合物的结构，并使用生物化学和生物物理方法来确定EspFu的多个序列重复之间的协同性是否来自WASP寡聚化。我们还将使用多学科的方法来确定寡聚化的相对重要性，并在WASP激活过程中由一组含SH 3的蛋白质缓解自抑制。我们已经重建了400 kDa的异源五聚体WAVE调节复合物（WRC）和来自人类和苍蝇的各种亚复合物，并将使用这些强大的试剂来了解WAVE如何受到上游输入（包括Rac和寡聚剂）的控制。这些调查将补充一个关键的三聚体亚复合物的WRC的结构测定。整个计划将揭示各种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，我们将开发一个新的概念，即WASP/WAVE活性是分层控制的;内层的调节控制非活性和活性状态之间的平衡，外层控制Arp 2/3复合物的活性状态的亲和力。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Arp 2/3复合物上产生复杂的肌动蛋白动力学。这项工作还将调和两种对立的WAVE监管模式，并揭示WASP/WAVE家族共同和独特的监管原则。我们的研究结果将解决生物物理学，信号转导和细胞生物学中的基本问题，并可能为癌症，人类遗传疾病和细菌感染的检测和治疗提出新的和改进的方法。我们的研究重点是了解连接Rho GTPases到WASP/WAVE蛋白到Arp 2/3复合物的信号通路。这些途径在许多正常的生物学过程和许多疾病中至关重要，包括转移性癌症、免疫疾病和细菌/病毒感染。了解这些蛋白质如何在这些途径中交流，以及如何被细菌转移，可能会导致诊断和治疗许多疾病的新药物。