Rab GTPases are essential regulators of diverse cellular processes including membrane trafficking, cell growth, organelle biogenesis, membrane remodeling, signaling transduction, and development. Rab GTPases as well as their regulatory factors and effectors have been implicated in genetically linked disorders, complex disease states such as type II diabetes and cancer, and are targets of virulence factors from pathogenic organisms. Many Rab GTPase have spatially and temporally overlapping distributions within the dynamic and highly interconnected network of tubular-vesicular organelles that comprise the biosynthetic, endocytic, and recycling systems. The functions of Rab GTPases are further coordinated through multivalent proteins and complexes with two or more distinct Rab GTPase binding domains, each of which has a unique specificity profile for subsets of Rab GTPases. With 60 distinct proteins encoded in the human genome, Rab GTPases represent the largest and most complex branch of the Ras superfamily. Characterizing interactions with structurally diverse effectors and regulatory factors, determining specificities, and deciphering the elaborate encoding of recognition determinants represent a critically important challenge. During the previous funding period, we developed, optimized, and successfully tested a multidisciplinary strategy for solving the recognition problem at the level of the Rab GTPase family. The long term objectives of this renewal application are to: i) generalize this approach to as many effectors, regulatory factors, and accessory proteins as possible;and ii) extend the methodology to incorporate identification of interaction partners for candidate Rab interacting proteins. To achieve these objectives, we will:
(Aim 1) quantitatively profile the specificity of effectors and regulatory factors for the Rab GTPase family;
(Aim 2) investigate the underlying structural bases;
(Aim 3) identify the major determinants of the observed specificity. This combination of experimental strategies will provide critical information on the specificity profiles, structural bases, and sequence determinants underlying the interaction of Rab GTPases with effectors, regulatory factors, and accessory proteins. Relevance to Public Health: These studies will reveal novel insights into the mechanisms for regulation of membrane trafficking, cell growth, and metabolism. The resulting information may prove useful in the design of mechanism based therapeutics for treatment of cancer and type II diabetes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Dunsmore, Sarah
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University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
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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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