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.
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.
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