Oxidative stress has been implicated in the pathogenesis of cancer, Alzheimer's, diabetic cataractogenesis, atherosclerosis, and alcoholic liver disease. Although the exact mechanism by which oxidative stress is involved in these processes is not entirely clear, evidence suggests that the lipid peroxidation product 4-hydroxynonenal (4-HNE) may be a specific causative agent. Because of its ability to covalently modify proteins, 4-HNE can theoretically interfere with signaling proteins, enzymes, and structural proteins. Because 4-HNE must interact with target biomolecules to cause deleterious effects, identification of these targets within the cell, and determining their physiological relevance is crucial. The first specific aim will be to characterize the interaction of 4-HNE with model proteins, defining the reaction kinetics between 4-HNE and protein. Immunoblot analysis will be used to verify the formation of adducts, and mass spectrometric techniques will be used to identify the number and sites of adduction. In the second specific aim, specific targets for 4-HNE adduction within the cell will be identified. This goal will be accomplished using cell culture models of 4-HNE exposure, with subsequent immunodetection of 4-HNE adducts. The identity of proteins adducted by 4-HNE will be identified using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis. Sites of 4-HNE adduction will be determined by PSD sequence analysis and adduct identification of in-gel generated tryptic fragments of the proteins. By identifying specific targets for 4-HNE adduction, the mechanism by which 4-HNE causes cellular dysfunction will be better understood. With this knowledge, strategies may be developed which are designed to protect from oxidative stress caused by increased generation of 4-HNE.
