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 GTP酶及其调节因子和效应物与遗传性疾病、复杂的疾病状态，如II型糖尿病和癌症有关，并且是病原体毒力因子的靶标。许多Rab GTP酶在动态和高度互联的管泡细胞器网络中具有空间和时间上的重叠分布，该网络组成了生物合成、内吞和循环系统。Rab GTP酶的功能进一步通过多价蛋白质和具有两个或更多不同的Rab GTP酶结合域的复合体来协调，每个结合域都有Rab GTP酶亚群的独特特异性。Rab GTP酶在人类基因组中编码60种不同的蛋白质，是RAS超家族中最大和最复杂的分支。表征与结构不同的效应器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精心编码，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，用于在RAB GTPase家族的水平上解决识别问题。这一更新应用的长期目标是：i)将这一方法推广到尽可能多的效应器、调节因子和辅助蛋白；以及ii)扩展方法学，包括识别候选RAb相互作用蛋白的相互作用伙伴。为了实现这些目标，我们将：(目标1)定量描述Rab GTPase家族的效应者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。这一实验策略的组合将提供关于Rab GTP酶与效应器、调节因子和辅助蛋白相互作用的特异性图谱、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、细胞生长和新陈代谢调控机制的新见解。由此得到的信息可能会被证明对癌症和II型糖尿病的基于机制的治疗方法的设计有用。

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.