Abstract
Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics, and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. It is likely that topological, chemical, and physical features combine to determine which proteins become targets for chemical adduction. Chemical-induced post-translational modification of certain critical proteins causes a change in structure/function that contributes to the toxicological response to chemical exposure. In this study, we have identified a number of proteins that are modified by quinone-thioethers after administration of 2-(glutathion-S-yl)HQ. Parallel one-dimensional gel electrophoresis was performed, and the Coomassie-stained gel was aligned with the corresponding Western blot, which was probed for adductions. Immunopositive bands were then subjected to trypsin digestion and analyzed via liquid chromatography/tandem mass spectrometry. The proteins that were subsequently identified contained a higher than average (9.7 versus 5.5%) lysine content and numerous stretches of lysine run-ons, which is a presumed electrophile binding motif. Approximately 50% of these proteins have also been identified as targets for electrophilic adduction by a diverse group of chemicals by other investigators, implying overlapping electrophile adductomes. By identifying a motif targeted by electrophiles it becomes possible to make predictions of proteins that may be targeted for adduction and possible sites on these proteins that are adducted. An understanding of proteins targeted for adduction is essential to unraveling the toxicity produced by these electrophiles.
Footnotes
-
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM070890]; and the National Institutes of Health National Institute of Environmental Health Sciences [Grants T32-ES07091, P30-ES006694, P30-ES007784].
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.108.026211.
-
ABBREVIATIONS: MS, mass spectrometry; HPLC, high-performance liquid chromatography; BQ, 1,4-benzoquinone; QT, quinol-thioether; EBM, electrophile-binding motif; MGHQ, 2-(glutathion-S-yl)-hydroquinone; 2-BrHQ-NAC, 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone; ECL, enhanced chemiluminescence; PBS, phosphate-buffered saline; γ-GT, γ-glutamyltranspeptidase; OSOM, outer stripe of the outer medulla; PAGE, polyacrylamide gel electrophoresis; MS/MS, tandem mass spectrometry; amu, atomic mass unit; NCBI, National Center for Biotechnology Information; KLH, keyhole-limpet hemocyanin; LC, liquid chromatography; MT, metallothionein; 4-HNE, 4-hydroxynonenal.
- Received December 23, 2008.
- Accepted February 23, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|