Within the broad goal of identifying biomarkers of human disease, the objective of the proposed studies is to define the chemical basis for the formation of endogenous DNA, RNA and protein adducts, with a focus on the roles of deoxyribose and lipid oxidation in generating these lesions. We propose to build on our observation of DNA and protein adducts derived from deoxyribose oxidation by moving the studies into living cells and tissues, by extending the studies to RNA as a potential target for reactive electrophiles, and by applying new technologies to study the DNA, RNA and protein adducts formed by analogous electrophiles arising from lipid peroxidation. Altogether, the four specific aims of the proposed studies should provide new insights into the chemical basis for endogenous nucleic acid and protein adducts that may be useful as biomarkers of exposure and disease.
Aim #1) Protein and DNA adducts derived from 4'-oxidation of deoxyribose. We will continue to study the role of base propenals and lipid peroxidation in the formation of the M1dG adduct in DNA and RNA in human cells, and expand 4'-oxidation adduct studies to include the 2-deoxypentose-4-ulose abasic site.
Aim 2) Protein and DNA adducts derived from 5'-oxidation of deoxyribose. We will move our studies of the novel DNA adducts derived from 2-phosphoryl-1,4-dioxobutane, a product of deoxyribose 5'-oxidation, into cells and tissues of animal models of human disease and expand the studies to include lysine adducts in histone proteins.
Aim 3) Histone adducts derived from 3'-formylphosphate residues. We will continue our studies of lysine N6- formylation in histones by proteomic mapping of the adduct locations, by LC/MS/MS quantification of adducts in cells and tissues, and by assessing the reactivity of histone deactylases with formyllysines in peptides.
Aim 4) Lipid peroxidation as a source of DNA, RNA and protein adducts. Given the parallels between deoxyribose and lipid oxidation, we propose to extend our deoxyribose studies by surveying the extent of protein, DNA and RNA adduct formation by lipid peroxidation products by incorporating 14C-labeled PUFA into cell membranes followed by quantification of 14C in protein, DNA and RNA fractions and identification of the DNA and RNA adducts. Subsequent efforts will be directed at defining structures of major adducts.
