Abstract
An electrophilic 5-methylene-2-pyrrolone modification (K(MP) ) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic K(MP) modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (K(Ac) ) modification but is more electrophilic. In this study we show that K(MP) -containing histone peptides are recognized by, and covalently modify bromodomain proteins that are K(Ac) readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with K(MP) -containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the K(Ac) binding site, indicating that K(MP) -containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of K(MP) could affect cellular function and viability by modifying proteins that regulate genetic expression.