Carbonyl stress by glyceraldehyde promotes glycation of nuclear non-muscle myosins in DNA–protein crosslink assemblies

DNA–protein crosslinks are bulky lesions that block DNA replication and transcription and are increasingly recognized in metabolic disorders characterized by carbonyl stress. Glyceraldehyde is a highly reactive triose that forms advanced glycation end-products, but its contribution to nuclear DNA–protein crosslink formation has not been defined. We examined glyceraldehyde-induced DNA–protein crosslinks in rat cardiomyoblast-derived H9c2 cells using an advanced recovery of potassium sodium dodecyl sulfate precipitates (ARK) assay combined with liquid chromatography–tandem mass spectrometry. Glyceraldehyde exposure modestly but significantly increased DNA–protein crosslink levels and generated nuclear high-molecular-weight complexes enriched in glycation adducts. Proteomic profiling of the DNA-tethered protein fraction revealed non-muscle myosin heavy chain isoforms 9 and 10 among the most abundant components. Liquid chromatography–tandem mass spectrometry mapping identified glycation adducts on multiple arginine residues within the coiled-coil rod and non-helical tail regions of non-muscle myosin heavy chains, including Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine and argpyrimidine. These modifications coincided with detergent-insoluble, high-molecular-weight myosin species in nuclear fractions. Our data identify glycated non-muscle myosins as major constituents of glyceraldehyde-induced, DNA-tethered DPC-associated assemblies and suggest a link between carbonyl stress and DNA damage associated with nuclear protein aggregation in cardiomyoblasts.