Rab, Arf and Arl GTPases function as core regulatory components of systems that control membrane trafficking as well as the biogenesis, identity, and maturation of membrane delimited organelles. Defects in these fundamental control mechanisms are implicated in complex disease states and genetically-linked disorders. The functional cycle and outputs of trafficking GTPases, including activation by guanine nucleotide exchange factors (GEFs), deactivation by GTPase activating proteins (GAPs), and interactions with effectors, are also manipulated by viral, bacterial, and eukaryotic pathogens to evade host responses and support replication. Understanding the mechanisms underlying trafficking regulation by GTPases and how these mechanisms are manipulated by pathogens and dysregulated in disease conditions represents a critically important challenge. Membrane recruitment and autoregulation of GEFs and GAPs are major paradigms for controlling the functional outputs of trafficking GTPases. The central objective of this application is to discern new principles underlying spatiotemporal control of GEF and GAP catalytic activities by combining multiple biochemical, biophysical, and structural approaches to investigate the molecular and structural mechanisms from host and pathogen perspectives. Specially, we will investigate structural mechanisms for: Arf GEF membrane recruitment and allosteric activation (Aim 1); membrane targeting of the Legionella pneumophila GEF DrrA and interaction with plasma membrane syntaxins (Aim 2); and Rab deactivation by GAPs and manipulation by pathogens. These studies will provide new insights into trafficking control mechanisms in host organisms and how these processes are subverted by intracellular pathogens. The results may be useful for the design of mechanism- based therapeutic strategies targeting GTPase dependent trafficking processes.

Public Health Relevance

Arf, Arl, and Rab GTPases are core control elements of systems that regulate the biogenesis and trafficking of cellular organelles. These GTPases, their effector proteins, and the enzymes that control their activation and deactivation are manipulated by viral, bacterial, and eukaryotic pathogens and dysregulated in complex disease states including diabetes, cancer, and neuropathies. The underlying mechanisms and disease etiologies remain poorly characterized. The research in this application will investigate molecular and structural bases for trafficking control by GTPases and how these fundamental regulatory processes are subverted by intracellular pathogens. The insights derived from these studies may be useful for development of novel mechanism-based therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056324-21
Application #
9471407
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Nie, Zhongzhen
Project Start
1998-05-01
Project End
2019-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
21
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
Malaby, Andrew W; Das, Sanchaita; Chakravarthy, Srinivas et al. (2018) Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors. Structure 26:106-117.e6
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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
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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

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