New Identified Posttranslational Modified (Glycated) Lysines and Arginines in Histones in Aging Human Heart/Endothelial Cells: Impact on Histone Octamer and Nucleosome Stability

Background: Posttranslational modifications (PTMs) of histones play an important role in epigenetic regulation and in the aging process. Nonenzymatic PTMs of lysines and arginines like advanced glycation end products may interfere with enzymatically controlled PTMs like acetylation and methylation. We therefore aimed to identify such modifications in aged human heart and aging primary human endothelial cells to identify their potential regulatory roles.

Method: Nuclear extracts of right human atrial appendages of young (<50 years) and old (>80 years) patients undergoing bypass surgery as well as low (young) and high (old) passaged HUVEC cells were analyzed by mass spectrometry to identify age-related PTMs/glycation sites. Site-directed mutagenesis was applied to simulate single identified hot-spots of glycation in histones at protein–protein or protein–DNA interaction sites. Recombinant and WT histones were expressed in E. coli and purified by ion-exchange and gel filtration chromatography. Octamer as well as nucleosome stability was analyzed by thermal shift assays in vitro.

Results: Ten identified glycation sites of lysines (carboxymethyl lysin, CML) and arginines (hydroimidazolone, MG-H1) in histones of aging senescent endothelial cells could be verified in right human atrial appendages in vivo. For four selected sites, site-directed mutagenesis (K > Q for CML, R > Y for MGH1) simulates charge and size of identified PTMs. WT and H2AK95Q, H2BK43Q, H3R43Y, and H4K31Q overexpressed and purified histones were used for octamer and nucleosome stability assays. In comparison to WT histones, H4K31Q leads to reduced thermal stability of the octamers, whereas H2BK43Q and H3R43Y decrease nucleosome stability.

Conclusion: The identified single nonenzymatic PTMs may have an impact on epigenetic regulation within the aging cardiovascular system by modifying histone as well as nucleosome stability. Further experiments like overexpression of WT/mutated histones in cells have to be done to understand their role in cellular function.

Publication History

Article published online:
28 January 2023