Proteomic Analysis of C. elegans Insulin Signaling. Abstract. Diabetes is a common metabolic disease frequently associated with defects in insulin signaling. The model organism C. elegans has emerged as an excellent system to study the insulin signaling pathway, which is highly conserved between humans and C. elegans. This proposal seeks to utilize mass spectrometry-based protein identification and quantification technologies developed in our lab to identify and characterize novel components of the C. elegans insulin signaling pathway. To find out if protein cofactors and post-translational modifications of basic components in this pathway help define a specific signaling outcome, tagged DAF-2 (C. elegans insulin receptor) and DAF-16 (FoxO forkhead transcription factor) will be purified from C. elegans and analyzed by mass spectrometry to identify these cofactors and modifications in relation to signaling conditions. Downstream targets of insulin signaling will be identified using 15N metabolic labeling and quantitative proteomic analyses of wild type, daf- 2 and daf-16 mutants. Novel insulin signaling components identified in this study will be inactivated using RNAi and C. elegans insulin signaling assays will be used to assess their physiological significance. Proteomic analysis of the insulin signaling pathway in conjunction with C. elegans genetics will lead to a better understanding of insulin signaling and provide new therapeutic targets for treatment of diabetes. Public Health Relevance: The body of knowledge produced by this proposal will give insights to the mechanism of insulin signaling and the underlying abnormalities in the diseased state. This can form the basis of future anti-diabetic treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK074798-03
Application #
7577347
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
Project Start
2007-03-01
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
3
Fiscal Year
2009
Total Cost
$371,070
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Fonslow, Bryan R; Stein, Benjamin D; Webb, Kristofor J et al. (2014) Addendum: Digestion and depletion of abundant proteins improves proteomic coverage. Nat Methods 11:347-8
Fonslow, Bryan R; Moresco, James J; Tu, Patricia G et al. (2014) Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes. WormBook :1-18
Fonslow, Bryan R; Stein, Benjamin D; Webb, Kristofor J et al. (2013) Digestion and depletion of abundant proteins improves proteomic coverage. Nat Methods 10:54-6
Zhang, Yaoyang; Fonslow, Bryan R; Shan, Bing et al. (2013) Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113:2343-94
Wang, Yueju; Fonslow, Bryan R; Wong, Catherine C L et al. (2012) Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis. Anal Chem 84:8505-13
Fonslow, Bryan R; Niessen, Sherry M; Singh, Meha et al. (2012) Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT. J Proteome Res 11:2697-709
Yates 3rd, John R; Park, Sung Kyu Robin; Delahunty, Claire M et al. (2012) Toward objective evaluation of proteomic algorithms. Nat Methods 9:455-6
Qu, Meng; Yang, Bing; Tao, Li et al. (2012) Phosphorylation-dependent interactions between Crb2 and Chk1 are essential for DNA damage checkpoint. PLoS Genet 8:e1002817
Fonslow, Bryan R; Carvalho, Paulo C; Academia, Katrina et al. (2011) Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT. J Proteome Res 10:3690-700
Fonslow, Bryan R; Kang, Seong A; Gestaut, Daniel R et al. (2010) Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes. Anal Chem 82:6643-51

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