Structural Study of GTPase Regulators and Effectors

描述(申请人提供)：Rho GTP酶CDC42和Rac通过WASP家族中的蛋白质向肌动蛋白细胞骨架发出信号。该奖项之前的研究发现，WASP自抑制结构域如何被Cdc42破坏稳定，导致细胞肌动蛋白核化机器Arp2/3复合体的激活。这里的研究将加深我们对WASP调控的理解，并解决生物物理学、信号转导和细胞生物学的重要新问题。已经发现了两种不相关的WASP抑制剂，它们似乎通过将WASP自抑制平衡偏置到非活性状态来发挥作用。我们将解开这些分子与WASP络合物的结构，并通过一系列生物物理分析，在定量水平上了解它们如何稳定自抑制结构域，阻断Cdc42和Arp2/3复合体的相互作用。我们将定量检测Cdc42对WASP的磷酸化和去磷酸化动力学的影响，以了解WASP是否能够感觉到GTP酶和激酶/磷酸酶信号的重合。我们将发现在Arp2/3实验中，磷酸化是否使WASP能够被SH2结构域激活，以及SH2结构域是否与已知的WASP激活剂协同作用。我们将使用对一系列不稳定的WASP蛋白进行的一系列生物物理/生化分析来关联WASP的稳定性、对CDC42的亲和力和对Arp2/3的活性，最终导致一个基于经典变构的WASP调控模型。最后，我们将利用核磁共振和生化来发现RAC的靶标WASP家族成员SCAR是如何被Pir121/NAP-1复合体抑制的，揭示了WASP家族调控的共同特征。这项工作将导致对WASP信号整合的两种机制的结构和热力学的理解，即作用于其自身抑制平衡的配体(蛋白质和小分子)的协同调节，以及偶发磷酸化。它将揭示自抑制的一般结构和热力学原理，以及以这种方式调节的分子如何在细胞中使用。联合计划将解决信号转导中的基本问题，并可能导致通过控制构象平衡发挥作用的新类型的细胞生物探针和药物制剂。这些分子在细胞骨架的研究和疾病的治疗中可能非常有价值，包括转移性癌症、免疫系统障碍和细菌/病毒感染。

DESCRIPTION (provided by applicant): The Rho GTPases Cdc42 and Rac signal to the actin cytoskeleton through proteins in the WASP family. Previous studies under the award discovered how the WASP autoinhibited domain is destabilized by Cdc42, leading to activation toward Arp2/3 complex, the cellular actin nucleation machine. Studies here will deepen our understanding of WASP regulation and address important new issues in biophysics, signal transduction and cell biology. Two unrelated WASP inhibitors have been discovered that appear to act by biasing the WASP autoinhibitory equilibrium to the inactive state. We will solve the structures of these molecules in complex with WASP, and learn at a quantitative level how they stabilize the autoinhibited domain, blocking Cdc42 and Arp2/3 complex interactions, through a series of biophysical assays. We will quantitate the effect of Cdc42 on phosphorylation and dephosphorylation kinetics of WASP, to learn if WASP could sense coincidence of GTPase and kinase/phosphatase signals. We will discover if phosphorylation enables WASP to be activated by SH2 domains in the Arp2/3 assay, and if SH2 domains act cooperatively with known WASP activators. We will use a battery of biophysical/ biochemical assays performed on a series of destabilized WASP proteins to correlate WASP stability, affinity for Cdc42 and activity toward Arp2/3, ultimately leading to a model for WASP regulation based on classical allostery. Finally, we will use NMR and biochemistry to discover how the WASP family member Scar, a target of Rac, is inhibited by the Pir121/NAP-1 complex, revealing common features of WASP family regulation. The work will lead to a structural and thermodynamic understanding of 2 mechanisms of signal integration by WASP, cooperative regulation by ligands (both protein and small molecule) that act on its autoinhibitory equilibrium, and contingent phosphorylation. It will reveal general structural and thermodynamic principles of autoinhibition, and how molecules regulated in this fashion may be used in the cell. The combined program will address fundamental issues in signal transduction, and could lead to new classes of cell biological probes and pharmaceutical agents that act through controlling conformational equilibria. Such molecules could be extremely valuable in studies of the cytoskeleton and treatment of diseases including metastatic cancer, immune system disorders and bacterial/viral infection.