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 N δ-(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.