Abstract 279 Posttranslational modified (glycated) lysins and arginins in core 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. Non-enzymatic PTMs of lysins and
arginins like Advanced Glycation Endproducts may interfere with enzymatically controlled PTMs like acetylation and methylation at the same sites. We, therefore,
aimed to identify such non-enzymatic modifications in aged human hearts and aging primary human endothelial cells to identify their potential regulatory roles.

Methods

Nuclear extracts of right human atrial appendages of young (<50 y) and old (>80 y) patients undergoing bypass surgery as well as of 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 conserved 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. HEK293 cells were
used for overexpression of histones.

Results
Ten identified glycation sites of lysins (Carboxymethyllysin, CML) and arginins (Hydroimidazolone, MG-H1) in histones of aging senescent endothelial cells could

be verified in right human atrial appendages in vivo. For 4 selected sites, site directed mutagenesis (K>Q for CML, R>Y for MGH1) simulate 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 as well as H3R43Y decrease nucleosome stability. First
results from overexpression of these modified histones in HEK293 cells indicate an impact on cell morphology and cellular senescence.

Conclusion
The identified single non-enzymatic PTMs may have an impact on epigenetic regulation within the aging cardiovascular system by modifying histone as well as

nucleosome stability. Further experiments with overexpression of WT/mutated histones in endothelial cells have to be done to understand their role in cellular function.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Posttranslational modified (glycated) lysins and arginins in core histones in aging human heart / endothelial cells: impact on histone octamer and nucleosome stability
S. Karande¹, A. Urazova¹, T. Gruber², J. Balbach², G. Szabó¹, A. Simm¹
¹Universitätsklinik und Poliklinik für Herzchirurgie, Universitätsklinikum Halle (Saale), Halle (Saale); ²Institut für Physik, Halle (Saale);
Background Posttranslational modifications (PTMs) of histones play an important role in epigenetic regulation and in the aging process. Non-enzymatic PTMs of lysins and arginins like Advanced Glycation Endproducts may interfere with enzymatically controlled PTMs like acetylation and methylation at the same sites. We, therefore, aimed to identify such non-enzymatic modifications in aged human hearts and aging primary human endothelial cells to identify their potential regulatory roles. Methods Nuclear extracts of right human atrial appendages of young (<50 y) and old (>80 y) patients undergoing bypass surgery as well as of 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 conserved 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. HEK293 cells were used for overexpression of histones. Results Ten identified glycation sites of lysins (Carboxymethyllysin, CML) and arginins (Hydroimidazolone, MG-H1) in histones of aging senescent endothelial cells could be verified in right human atrial appendages in vivo. For 4 selected sites, site directed mutagenesis (K>Q for CML, R>Y for MGH1) simulate 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 as well as H3R43Y decrease nucleosome stability. First results from overexpression of these modified histones in HEK293 cells indicate an impact on cell morphology and cellular senescence. Conclusion The identified single non-enzymatic PTMs may have an impact on epigenetic regulation within the aging cardiovascular system by modifying histone as well as nucleosome stability. Further experiments with overexpression of WT/mutated histones in endothelial cells have to be done to understand their role in cellular function.
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