Structural basis for Rab GTPase regulated membrane trafficking

描述(由申请人提供)：RAB GTP酶是真核细胞膜成熟和跨膜系统运输的主要调节因子。Rab GTP酶的功能依赖于‘Inactive’(GDP结合)和‘Active’(GTP结合)状态之间的构象转换周期。激活和失活是由GDP/GTP交换因子(GEF)和GTP酶激活蛋白(GAP)严格控制的，它们加速了固有的缓慢的核苷酸交换和GTP水解率。活性Rab GTP酶与参与膜转运所有阶段的各种效应器相互作用。活性Rab GTP酶的功能输出通过具有多个Rab结合位点的多价效应器整合。活性的Rab GTP酶还为下游或上游的Rab GTP酶招募GEF或GAP，以协调连续的转运阶段，促进膜成熟。这一建议的总体目标是了解Rab GEF和GAP膜招募的结构和分子基础，招募后Rab GTP酶的选择性激活和失活，以及微生物病原体对这些宿主过程的操纵。具体地说，我们将结合结晶学、突变和基于细胞的分析与定量的Rab家族识别图谱来研究：(Aim 1)Denn和Vps9结构域GEF的招募和Rab识别/激活；(Aim 2)TBC结构域间隙的招募和Rab识别/激活；(Aim 3)嗜肺军团菌对宿主Rab GTP酶的操纵。这些目标的成功完成将为在正常和致病条件下支持Rab GTPase调控的膜生物学的特征不佳的分子相互作用网络和结构机制提供新的见解。 公共卫生相关性：RAB GTP酶及其GEF、GAP和效应因子与各种致病因素有关，包括遗传连锁疾病、病毒和细菌感染以及糖尿病和癌症等复杂疾病。潜在的病因在很大程度上是不被理解的，因此在制定治疗干预策略方面也没有得到充分的利用。这项建议中概述的研究将调查Rab GTP酶调控的膜生物学的一些最不完善的机制，以及这些基本的细胞过程如何被导致军团病的细胞内病原体颠覆。

DESCRIPTION (provided by applicant): Rab GTPases are key master regulators of membrane maturation and trafficking throughout the endomembrane system of eukaryotic cells. The function of Rab GTPases depends on a conformational switch cycle between 'inactive' (GDP-bound) and 'active' (GTP-bound) states. Activation and deactivation are tightly controlled by GDP/GTP exchange factors (GEFs) and GTPase activating proteins (GAPs) that accelerate the intrinsically slow rates of nucleotide exchange and GTP hydrolysis. Active Rab GTPases interact with diverse effectors involved in all stages of membrane trafficking. The functional outputs of active Rab GTPases are integrated through multivalent effectors with multiple Rab binding sites. Active Rab GTPases also recruit GEFs or GAPs for downstream or upstream Rab GTPases to coordinate successive trafficking stages and facilitate membrane maturation. The overall goal of this proposal is to understand the structural and molecular bases underlying membrane recruitment of Rab GEFs and GAPs, selective activation and deactivation of Rab GTPases following recruitment, and manipulation of these host process by microbial pathogens. Specifically, we will combine crystallographic, mutational and cell based analyses with quantitative Rab family recognition profiles to investigate the structural and molecular bases for: (Aim 1) recruitment and Rab recognition/activation by DENN and Vps9 domain GEFs; (Aim 2) recruitment and Rab recognition/activation by TBC domain GAPs; (Aim 3) manipulation of host Rab GTPases by Legionella pneumophila. Successful completion of these aims will deliver new insights into poorly characterized molecular interaction networks and structural mechanisms that underpin Rab GTPase-regulated membrane biology in normal and pathogenic conditions. PUBLIC HEALTH RELEVANCE: Rab GTPases and their GEFs, GAPs and effectors have been implicated in a variety of pathogenic conditions including genetically linked disorders, viral and bacterial infections, and complex diseases such as diabetes mellitus and cancer. The underlying etiology is for the most part poorly understood and consequently underexploited in the development of strategies for therapeutic intervention. This research outlined in this proposal will investigate some of the least well characterized mechanisms of Rab GTPase-regulated membrane biology and how these fundamental cellular processes can be subverted by the intracellular pathogen responsible for Legionaire's disease.