Reactive electrophiles generated by environmental agents and stresses can modify proteins and trigger adaptive cellular responses reflected by gene expression and protein functional changes. Cysteine thiol groups are thought to be key sites of important oxidative or covalent adduct modification, but the specific structural characteristics that cast proteins in sentinel roles are unknown. The overall objective of this project is to identify specific protein targets that serve as triggers for cellular stress responses. We hypothesize that specific structural features allow some alkylation sensitive proteins to act as """"""""sensor-triggers"""""""" that readily react with electrophiles and that adduction alters critical protein-protein interactions, thus causing changes in kinase signaling and transcription factor regulation. We will use new proteomics liquid chromatography-tandem mass spectrometry (LC-MS-MS) tools and approaches to study the mechanisms by which protein modifications alter key protein-protein interactions that regulate stress responses.
The specific aims of the proposed research are: 1) To identify protein classes, sequence motifs and domain structures that are targets for thiol-reactive electrophiles in human cells. Biotin-linked electrophiles will be used to combine affinity enrichment and new LC-MS-MS approaches to identify modified proteins and map the adducts to specific sequences. Different thiol-reactive electrophile chemistries will be used to establish the generality of the targets identified. Bioinformatics tools will be used to associate adducted sequences with specific domain structures and motifs. 2) To validate specific proteins as targets for thiol-reactive electrophiles in intact cells. Proteins identified as targets for biotin-linked electrophiles in Specific Aim 1 will be evaluated as targets for model electrophiles that correspond to their biotin-linked counterparts in cell models. 3) To characterize the effects of protein adduction on selected protein enzymatic activities and on selected protein-protein interactions in reconstituted systems in vitro. Quantitative proteomics methods will be applied to compare adduction with changes in target activities and protein-protein interactions in a parallel in vitro model. These studies will lay the groundwork for applying molecular target analysis to proteomes in toxicology.
| Michaelson-Richie, Erin D; Ming, Xun; Codreanu, Simona G et al. (2011) Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. J Proteome Res 10:2785-96 |
| Michaelson-Richie, Erin D; Loeber, Rachel L; Codreanu, Simona G et al. (2010) DNA-protein cross-linking by 1,2,3,4-diepoxybutane. J Proteome Res 9:4356-67 |
| Loeber, Rachel L; Michaelson-Richie, Erin D; Codreanu, Simona G et al. (2009) Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards. Chem Res Toxicol 22:1151-62 |
| Wong, Hansen L; Liebler, Daniel C (2008) Mitochondrial protein targets of thiol-reactive electrophiles. Chem Res Toxicol 21:796-804 |
| Rachakonda, Girish; Xiong, Ying; Sekhar, Konjeti R et al. (2008) Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3. Chem Res Toxicol 21:705-10 |
| Liebler, Daniel C (2008) Protein damage by reactive electrophiles: targets and consequences. Chem Res Toxicol 21:117-28 |
| Lin, De; Saleh, Samir; Liebler, Daniel C (2008) Reversibility of covalent electrophile-protein adducts and chemical toxicity. Chem Res Toxicol 21:2361-9 |
| Lin, De; Saleh, Samir; Liebler, Daniel (2008) Reversibility of Covalent Electrophile-Protein Adducts and Chemical Toxicity. Chem Res Toxicol : |
| Shin, Nah-Young; Liu, Qinfeng; Stamer, Sheryl L et al. (2007) Protein targets of reactive electrophiles in human liver microsomes. Chem Res Toxicol 20:859-67 |
| Orton, Christopher R; Liebler, Daniel C (2007) Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles. Chem Biol Interact 168:117-27 |
Showing the most recent 10 out of 31 publications
