Structural basis for Rab GTPase regulated membrane trafficking

描述（由申请人提供）：Rab GTPases是真核细胞整个膜系统中膜成熟和运输的关键主调节剂。Rab GTPases的功能取决于“非活性”（gdp结合）和“活性”（gtp结合）状态之间的构象切换循环。激活和失活受到GDP/GTP交换因子（GEFs）和GTP酶激活蛋白（gap）的严格控制，它们加速了本质上缓慢的核苷酸交换和GTP水解速率。活性Rab gtpase与参与膜运输各个阶段的各种效应物相互作用。活性Rab gtpase的功能输出通过具有多个Rab结合位点的多价效应物进行整合。活性的Rab GTPases也为下游或上游的Rab GTPases招募gef或gap，以协调连续的运输阶段并促进膜成熟。本研究的总体目标是了解Rab gef和gap在膜上募集的结构和分子基础，募集后Rab gtpase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。具体来说，我们将结合晶体学、突变和基于细胞的分析与定量Rab家族识别谱来研究结构和分子基础：（目标1）DENN和Vps9结构域gef的招募和Rab识别/激活；（目标2）TBC结构域gap的招募和Rab识别/激活；（目的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调节的膜生物学的分子相互作用网络和结构机制提供新的见解。

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.