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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM056324-17
Application #
8652320
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Dunsmore, Sarah
Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
Worcester
State
MA
Country
United States
Zip Code
01655
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
Chotard, Laetitia; Mishra, Ashwini K; Sylvain, Marc-Andre et al. (2010) TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans. Mol Biol Cell 21:2285-96
Fang, Zhiyou; Takizawa, Norio; Wilson, Korey A et al. (2010) The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility. Traffic 11:782-99
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
Barr, Francis; Lambright, David G (2010) Rab GEFs and GAPs. Curr Opin Cell Biol 22:461-70
Lee, Meng-Tse Gabe; Mishra, Ashwini; Lambright, David G (2009) Structural mechanisms for regulation of membrane traffic by rab GTPases. Traffic 10:1377-89
Mukhopadhyay, Arnab; Pan, Xiaojing; Lambright, David G et al. (2007) An endocytic pathway as a target of tubby for regulation of fat storage. EMBO Rep 8:931-8
Delprato, Anna; Lambright, David G (2007) Structural basis for Rab GTPase activation by VPS9 domain exchange factors. Nat Struct Mol Biol 14:406-12

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