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.
|Mishra, Ashwini K; Lambright, David G (2016) Invited review: Small GTPases and their GAPs. Biopolymers 105:431-48|
|Malaby, Andrew W; Chakravarthy, Srinivas; Irving, Thomas C et al. (2015) Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering. J Appl Crystallogr 48:1102-1113|
|Kahn, Richard A; Lambright, David G (2015) A PH Domain with Dual Phospholipid Binding Sites Regulates the ARF GAP, ASAP1. Structure 23:1971-3|
|Priya, Amulya; Kalaidzidis, Inna V; Kalaidzidis, Yannis et al. (2015) Molecular insights into Rab7-mediated endosomal recruitment of core retromer: deciphering the role of Vps26 and Vps35. Traffic 16:68-84|
|Mishra, Ashwini K; Lambright, David G (2015) High-throughput assay for profiling the substrate specificity of Rab GTPase-activating proteins. Methods Mol Biol 1298:47-60|
|Del Campo, Claudia M; Mishra, Ashwini K; Wang, Yu-Hsiu et al. (2014) Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/SidM. Structure 22:397-408|
|Mishra, Ashwini K; Del Campo, Claudia M; Collins, Robert E et al. (2013) The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism. J Biol Chem 288:24000-11|
|Malaby, Andrew W; van den Berg, Bert; Lambright, David G (2013) Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors. Proc Natl Acad Sci U S A 110:14213-8|
|Davey, Jonathan R; Humphrey, Sean J; Junutula, Jagath R et al. (2012) TBC1D13 is a RAB35 specific GAP that plays an important role in GLUT4 trafficking in adipocytes. Traffic 13:1429-41|
|Mishra, Ashwini; Eathiraj, Sudharshan; Corvera, Silvia et al. (2010) Structural basis for Rab GTPase recognition and endosome tethering by the C2H2 zinc finger of Early Endosomal Autoantigen 1 (EEA1). Proc Natl Acad Sci U S A 107:10866-71|
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