This is an application for continuation of a long-term project to comprehensively identify and functionally analyze protein interactions and posttranslational modifications regulating to eukaryotic protein synthesis. Protein synthesis is a fundamental process essential for replication, growth, and maintenance of all living organisms. Aberrant protein synthesis contributes to a number of human diseases including cancer and mental retardation. Hence a comprehensive understanding of protein synthesis is needed for systems analysis of both the normal and disease-altered translation process. Our strategy combines tandem mass spectrometry-based proteomics with genetic and biochemical functional assays to identify and dissect the function of uncharacterized proteins and posttranslational modifications controlling translation. Initially, we identified and validated a large number of uncharacterized proteins and posttranslational modifications associated with ribosomes, polysomes, and translation factors in S. cerevisiae. In this application, experiments are proposed to determine the function of a subset of these proteins linked to translation initiation. Initiation is the main step in which translation is regulated. First, we will assay yeast deletion mutants using sensitive biochemical and genetic experiments to identify defects in translation initiation. To complement the genetic and biochemical assays, we will perform synthetic genetic analysis on a genome- wide scale to genetically identify interacting proteins. By combining these approaches, we expect to develop functional models for many of uncharacterized proteins. Second, we will identify selected posttranslational modifications on translation initiation factors. Mass spectrometry will be used to specifically identify the modified amino acids. These amino acids will be mutated to establish their biological significance. Finally, we will extend our interest in identifying translation initiation factors to the poorly understood process of cap- independent initiation. The essential cell-cycle gene ornithine decarboxylase (ODC) and the oncogene c- myc are translated through a cap-independent mechanism using internal ribosomal entry sites (IRES). We will use novel proteomic screens to identify proteins that bind specifically to these mRNA sites. Reporter assays will be used be to test candidate proteins for positive and negative effects on ODC and c-myc cap- independent translation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064779-09
Application #
7822797
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Bender, Michael T
Project Start
2002-02-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
9
Fiscal Year
2010
Total Cost
$334,323
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Howard, Leigh M; Hoek, Kristen L; Goll, Johannes B et al. (2017) Cell-Based Systems Biology Analysis of Human AS03-Adjuvanted H5N1 Avian Influenza Vaccine Responses: A Phase I Randomized Controlled Trial. PLoS One 12:e0167488
Galassie, Allison C; Goll, Johannes B; Samir, Parimal et al. (2017) Proteomics show antigen presentation processes in human immune cells after AS03-H5N1 vaccination. Proteomics 17:
Liang, Xijun; Xia, Zhonghang; Jian, Ling et al. (2015) An adaptive classification model for peptide identification. BMC Genomics 16 Suppl 11:S1
Galassie, Allison C; Link, Andrew J (2015) Proteomic contributions to our understanding of vaccine and immune responses. Proteomics Clin Appl 9:972-89
Hoek, Kristen L; Samir, Parimal; Howard, Leigh M et al. (2015) A cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination. PLoS One 10:e0118528
Samir, Parimal; Rahul; Slaughter, James C et al. (2015) Environmental Interactions and Epistasis Are Revealed in the Proteomic Responses to Complex Stimuli. PLoS One 10:e0134099
Jian, Ling; Niu, Xinnan; Xia, Zhonghang et al. (2013) A novel algorithm for validating peptide identification from a shotgun proteomics search engine. J Proteome Res 12:1108-19
Browne, Christopher M; Samir, Parimal; Fites, J Scott et al. (2013) The yeast eukaryotic translation initiation factor 2B translation initiation complex interacts with the fatty acid synthesis enzyme YBR159W and endoplasmic reticulum membranes. Mol Cell Biol 33:1041-56
Marionneau, Celine; Carrasquillo, Yarimar; Norris, Aaron J et al. (2012) The sodium channel accessory subunit Navýý1 regulates neuronal excitability through modulation of repolarizing voltage-gated Kýýý channels. J Neurosci 32:5716-27
Mushrush, Darren J; Koteiche, Hanane A; Sammons, Morgan A et al. (2011) Studies of the mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes. J Biol Chem 286:27011-8

Showing the most recent 10 out of 43 publications