Structural Study of GTPase Regulators and Effectors

描述（由申请人提供）：肌动蛋白细胞骨架的重排对许多细胞过程至关重要，并且在许多遗传和感染性疾病中存在缺陷。Wiskott-Aldrich综合征蛋白（WASP）家族成员在整个生物学过程中控制肌动蛋白动力学起关键作用。在之前的研究中，我们发现了WASP家族调控的一种新机制，它将大量不同的无法解释的数据统一在一个共同的框架下。我们还重组了两个五聚体，400 kDa的WRC和550 kDa的SHRC，它们分别含有和控制WASP蛋白WAVE和WASH。我们的WRC晶体结构解释了组装中WAVE的抑制作用。我们的生化分析解决了关于WRC活性的长期争议。在这里，我们将利用我们对重组WRC和SHRC的独特途径，从结构和生化角度了解这些组合如何以复杂的方式响应上游刺激，促进细胞迁移、神经元粘附和囊泡运输。我们将确定与Rac GTPase结合的WRC的晶体结构。这种结构，加上互补的生化和协同细胞生物学研究，将解释WRC是如何被gtp酶、磷脂和激酶协同激活的。我们将描述一种新的wrc结合基序（WIPS），我们已经在30个神经元粘附受体中发现，包括许多神秘的原钙粘蛋白家族成员。我们将确定WRC- wips复合物的结构，并了解各种含wips的受体如何在体外和细胞中与Rac合作激活WRC。最后，我们将了解SHRC如何通过与逆转录物外壳复合物的多价结合而被招募到膜上，以及这些相互作用对肌动蛋白组装的功能后果。我们的工作将允许首次在单链WASP蛋白（WASP/N-WASP）和多组分组件（所有其他家族成员）中功能的WASP蛋白之间进行物理比较，揭示跨家族信号整合的新一般原则。我们将学习原钙粘蛋白和其他神经元受体如何与肌动蛋白沟通，并与其他信号输入协调，作为其鲜为人知的粘附功能的一部分。我们的发现将提供新的试剂和概念来指导神经科学家理解细胞和生物体中的这些受体。我们将获得新的见解，包括自闭症，癫痫和耳聋，这可能是由原钙粘蛋白突变引起的疾病。最后，我们对SHRC的研究将通过一个新的假设来解决细胞生物学中一个广泛的重要问题——肌动蛋白组装和囊泡外壳形成在内噬运输过程中是如何协调的，即多价性提供了SHRC招募的机制，以响应膜上的反转录物密度。这项工作将提出一般的机制，通过多价相互作用可以用来控制可溶性物种的膜相互作用的特异性和时间。

DESCRIPTION (provided by applicant): Rearrangements of the actin cytoskeleton are critical to numerous cellular processes and are defective in many genetic and infectious diseases. Members of the Wiskott-Aldrich Syndrome Protein (WASP) family play key roles in controlling actin dynamics throughout biology. In the previous period we discovered a new mechanism of WASP family regulation that unified a disparate body of unexplained data under a common framework. We also reconstituted two pentameric assemblies, the 400 kDa WRC and the 550 kDa SHRC, that contain and control the WASP proteins WAVE and WASH, respectively. Our WRC crystal structure explained inhibition of WAVE within the assembly. Our biochemical analyses resolved a long-standing dispute regarding WRC activity. Here, we will exploit our unique access to recombinant WRC and SHRC to understand structurally and biochemically how these assemblies respond in complex fashion to upstream stimuli to promote cell migration, neuronal adhesion and vesicle trafficking. We will determine the crystal structure of the WRC bound to the Rac GTPase. The structure, plus complementary biochemical and collaborative cell biological studies, will explain how the WRC is cooperatively activated by GTPases, phospholipids and kinases. We will characterize a novel WRC-binding motif (WIPS) that we have discovered in 30 neuronal adhesion receptors, including many members of the enigmatic protocadherin family. We will determine the structure of a WRC-WIPS complex and learn how various WIPS-containing receptors cooperate with Rac to activate the WRC in vitro and in cells. Finally, we will learn how the SHRC is recruited to membranes through multivalent binding to the retromer coat complex, and the functional consequences of these interactions on actin assembly. Our work will allow the first physical comparisons between WASP proteins that function as single chains (WASP/N-WASP) and those that function within multi-component assemblies (all other family members), revealing new and general principles of signal integration that span the family. We will learn how protocadherins and other neuronal receptors communicate to actin and are coordinated with other signaling inputs as part of their poorly understood adhesive functions. Our findings will provide new reagents and concepts to guide neuroscientists in understanding these receptors in cels and organisms. We wil gain new insights into diseases including autism, epilepsy and deafness, which can be caused by protocadherin mutations. Finally, our studies of the SHRC will address a broadly significant problem in cell biology--how actin assembly and vesicle coat formation are cordinated during endocytic traficking-through a new hypothesis, that multivalency provides a mechanism for SHRC recruitment to respond to retromer density on membranes. This work will suggest general mechanisms by which multivalent interactions can be used to control the specificity and timing of membrane interactions of soluble species. PUBLIC HEALTH RELEVANCE: Our research focuses on understanding control of actin dynamics by members of the Wiskott-Aldrich Syndrome Protein (WASP) family. These molecules are critically involved in many normal biological processes and in numerous diseases, including metastatic cancer, immune disorders, infection, and as suggested by our recent findings, neuronal diseases including autism, epilepsy, deafness and blindness. An understanding of how WASP proteins function will reveal new, general principles in basic biology and could lead to new agents for the diagnosis and treatment of many diseases.