Structural basis for Rab GTPase regulated membrane trafficking

描述（由申请人提供）：Rab GTP酶是多种细胞过程的基本调节剂，包括膜运输、细胞生长、细胞器生物发生、膜重塑、信号转导和发育。Rab GTP酶以及它们的调节因子和效应物已经涉及遗传相关的病症、复杂的疾病状态如II型糖尿病和癌症，并且是来自病原生物体的毒力因子的靶标。许多Rab GTdR在管状泡状细胞器的动态和高度互连的网络内具有空间和时间上重叠的分布，所述管状泡状细胞器包括生物合成、内吞和再循环系统。Rab GTP酶的功能通过多价蛋白质和具有两个或更多个不同Rab GTP酶结合结构域的复合物进一步协调，每个结合结构域对Rab GTP酶的子集具有独特的特异性谱。在人类基因组中编码60种不同的蛋白质，Rab GTP酶代表Ras超家族中最大和最复杂的分支。表征与结构多样的效应物和调节因子的相互作用，确定特异性，并破译识别决定因素的精心编码是一个至关重要的挑战。在上一个资助期内，我们开发、优化并成功测试了一种多学科策略，用于解决Rab GTdash系列的识别问题。该更新申请的长期目标是：i）将该方法推广到尽可能多的效应物、调节因子和辅助蛋白;和ii）扩展该方法以并入候选Rab相互作用蛋白的相互作用伴侣的鉴定。为了实现这些目标，我们将：（目的1）定量分析Rab GT3家族的效应子和调节因子的特异性;（目的2）研究潜在的结构基础;（目的3）确定观察到的特异性的主要决定因素。这种实验策略的组合将提供关键信息的特异性配置文件，结构基础和序列决定因素的相互作用的Rab GTP酶与效应，调节因子和辅助蛋白。与公共卫生的相关性：这些研究将揭示膜运输，细胞生长和代谢的调节机制的新见解。由此产生的信息可能会证明有用的机制为基础的治疗癌症和II型糖尿病的治疗设计。

DESCRIPTION (provided by applicant): Rab GTPases are essential regulators of diverse cellular processes including membrane trafficking, cell growth, organelle biogenesis, membrane remodeling, signaling transduction, and development. Rab GTPases as well as their regulatory factors and effectors have been implicated in genetically linked disorders, complex disease states such as type II diabetes and cancer, and are targets of virulence factors from pathogenic organisms. Many Rab GTPase have spatially and temporally overlapping distributions within the dynamic and highly interconnected network of tubular-vesicular organelles that comprise the biosynthetic, endocytic, and recycling systems. The functions of Rab GTPases are further coordinated through multivalent proteins and complexes with two or more distinct Rab GTPase binding domains, each of which has a unique specificity profile for subsets of Rab GTPases. With 60 distinct proteins encoded in the human genome, Rab GTPases represent the largest and most complex branch of the Ras superfamily. Characterizing interactions with structurally diverse effectors and regulatory factors, determining specificities, and deciphering the elaborate encoding of recognition determinants represent a critically important challenge. During the previous funding period, we developed, optimized, and successfully tested a multidisciplinary strategy for solving the recognition problem at the level of the Rab GTPase family. The long term objectives of this renewal application are to: i) generalize this approach to as many effectors, regulatory factors, and accessory proteins as possible; and ii) extend the methodology to incorporate identification of interaction partners for candidate Rab interacting proteins. To achieve these objectives, we will: (Aim 1) quantitatively profile the specificity of effectors and regulatory factors for the Rab GTPase family; (Aim 2) investigate the underlying structural bases; (Aim 3) identify the major determinants of the observed specificity. This combination of experimental strategies will provide critical information on the specificity profiles, structural bases, and sequence determinants underlying the interaction of Rab GTPases with effectors, regulatory factors, and accessory proteins. Relevance to Public Health: These studies will reveal novel insights into the mechanisms for regulation of membrane trafficking, cell growth, and metabolism. The resulting information may prove useful in the design of mechanism based therapeutics for treatment of cancer and type II diabetes.