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是许多细胞过程的关键，在许多遗传和传染病中是有缺陷的。Wiskott-Aldrich综合征蛋白(WASP)家族的成员在控制整个生物学中的肌动蛋白动态方面发挥着关键作用。在前一个时期，我们发现了一种新的WASP家族调控机制，它将一系列不同的未解释数据统一在一个共同的框架下。我们还重组了两个五聚体组件，400 kDa WRC和550 kDa SHRC，它们分别包含和控制WASP蛋白的WAVE和WASH。我们的WRC晶体结构解释了组装内波的抑制。我们的生化分析解决了关于WRC活动的长期争议。在这里，我们将利用我们获得重组WRC和SHRC的独特途径，从结构和生化上了解这些组件如何以复杂的方式对上游刺激做出反应，以促进细胞迁移、神经元黏附和囊泡运输。我们将确定与RAC GTP酶结合的WRC的晶体结构。这种结构，加上互补的生化和协作细胞生物学研究，将解释WRC是如何被GTP酶、磷脂和激酶协同激活的。我们将描述我们在30个神经元黏附受体中发现的一个新的WRC结合基序(WIPS)，其中包括神秘的原钙粘附素家族的许多成员。我们将确定WRC-WIPS复合体的结构，并了解各种含有WIPS的受体如何在体外和细胞内与RAC合作激活WRC。最后，我们将了解SHRC是如何通过多价结合到逆转录被膜复合体上的，以及这些相互作用对肌动蛋白组装的功能影响。我们的工作将允许首次对作为单链功能的WASP蛋白质(WASP/N-WASP)和那些在多组分组件中发挥功能的蛋白质(所有其他家族成员)进行物理比较，揭示跨越家族的新的一般信号整合原则。我们将学习原钙粘附素和其他神经元受体如何与肌动蛋白通信，以及如何与其他信号输入协调，作为其鲜为人知的粘连功能的一部分。我们的发现将提供新的试剂和概念，指导神经科学家理解细胞和生物体中的这些受体。我们将对包括自闭症、癫痫和耳聋在内的疾病有新的见解，这些疾病可能是由原钙粘附素突变引起的。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意义上的问题--肌动蛋白组装和囊泡外壳的形成是如何协调的，即多价性为SHRC的招募提供了一种机制，以响应膜上的逆转录病毒密度。这项工作将提出一般的机制，通过这些机制可以利用多价相互作用来控制可溶性物种的膜相互作用的特异性和时间。 公共卫生相关性：我们的研究重点是了解Wiskott-Aldrich综合征蛋白(WASP)家族成员对肌动蛋白动态的控制。这些分子与许多正常的生物过程和许多疾病密切相关，包括转移性癌症、免疫紊乱、感染，以及我们最近发现的神经元疾病，包括自闭症、癫痫、耳聋和失明。对WASP蛋白质功能的了解将揭示基础生物学中新的一般原理，并可能导致许多疾病的诊断和治疗的新试剂。

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.