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重要调节因子，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 gtpase及其调控因子和效应物与遗传相关疾病、复杂疾病状态（如II型糖尿病和癌症）有关，并且是病原生物毒力因子的靶标。许多Rab GTPase在包括生物合成、内吞和循环系统的管泡状细胞器的动态和高度互联网络中具有空间和时间重叠分布。Rab GTPase的功能通过多价蛋白和具有两个或更多不同Rab GTPase结合结构域的复合物进一步协调，每个结构域对Rab GTPase亚群具有独特的特异性。在人类基因组中编码有60种不同的蛋白质，Rab GTPases代表了Ras超家族中最大和最复杂的分支。表征与结构多样的效应因子和调控因子的相互作用，确定特异性，并破译识别决定因素的复杂编码是一个至关重要的挑战。在之前的资助期间，我们开发、优化并成功测试了一种多学科策略，用于解决Rab GTPase家族水平的识别问题。这项更新申请的长期目标是：i)将这种方法推广到尽可能多的效应器、调节因子和辅助蛋白；ii)扩展方法，纳入候选Rab相互作用蛋白的相互作用伙伴的鉴定。为了实现这些目标，我们将：（目标1）定量分析Rab GTPase家族的效应物和调控因子的特异性；（目标2）调查潜在的结构基础；（目标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于Rab GTPases与效应物、调节因子和辅助蛋白相互作用的特异性特征、结构基础和序列决定因素的关键信息。与公共卫生相关：这些研究将揭示膜运输、细胞生长和代谢调节机制的新见解。所得到的信息可能在设计基于机制的治疗癌症和II型糖尿病的方法中被证明是有用的。

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.