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Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6)-formyllysine that is refractory to histone deacetylases.

Edrissi B, Taghizadeh K, Dedon PC - PLoS Genet. (2013)

Bottom Line: While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources.Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct.These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6)-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6)-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6)-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6)-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4) lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4) lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

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Summary of findings on N6-formyllysine in histones.N6-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N6-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.
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pgen-1003328-g007: Summary of findings on N6-formyllysine in histones.N6-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N6-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.

Mentions: The relative abundance of N6-formyllysine in histone and other proteins (Table 1 and Table 2) [9]–[11] and the persistence of these adducts in histone proteins provides insights into both their source and their potential effects on cell function. The N6-formyllysine residues are relatively evenly distributed among different classes of histone proteins (Table 1), while the other functional modifications show very biased distributions over a large frequency range, which is consistent with the known function and conserved locations for lysine methylation and acetylation [16]–[18]. This random distribution of formyllysine adducts in histone proteins suggests that they are adventitious and not physiological. The fact that N6-formyllysine levels are similar in histone and non-nuclear proteins and in all cell compartments also suggests that the sources of this protein modification are equally balanced in the various compartments and proteins, or that there is a single dominant source that distributes uniformly throughout the cell. With regard to its persistence in cells, there is still no evidence supporting the enzymatic removal of N6-formyllysine. Our investigation reveals that N6-formyllysine adducts appear to be refractory to removal by histone deacetylase enzymes, which suggests that they will persist throughout the lifetime of individual histone proteins. Although our results are consistent with lysine N6-formylation as a stable protein modification, we cannot rule out the possibility of an enzyme that removes this modification from selected conserved lysine sites in histone proteins, resulting in small changes in the population level of formyllysine that are not detectable by our current analytical method. Figure 7 summarizes our findings presented here on N6-formyllysine adducts.


Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6)-formyllysine that is refractory to histone deacetylases.

Edrissi B, Taghizadeh K, Dedon PC - PLoS Genet. (2013)

Summary of findings on N6-formyllysine in histones.N6-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N6-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585032&req=5

pgen-1003328-g007: Summary of findings on N6-formyllysine in histones.N6-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N6-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.
Mentions: The relative abundance of N6-formyllysine in histone and other proteins (Table 1 and Table 2) [9]–[11] and the persistence of these adducts in histone proteins provides insights into both their source and their potential effects on cell function. The N6-formyllysine residues are relatively evenly distributed among different classes of histone proteins (Table 1), while the other functional modifications show very biased distributions over a large frequency range, which is consistent with the known function and conserved locations for lysine methylation and acetylation [16]–[18]. This random distribution of formyllysine adducts in histone proteins suggests that they are adventitious and not physiological. The fact that N6-formyllysine levels are similar in histone and non-nuclear proteins and in all cell compartments also suggests that the sources of this protein modification are equally balanced in the various compartments and proteins, or that there is a single dominant source that distributes uniformly throughout the cell. With regard to its persistence in cells, there is still no evidence supporting the enzymatic removal of N6-formyllysine. Our investigation reveals that N6-formyllysine adducts appear to be refractory to removal by histone deacetylase enzymes, which suggests that they will persist throughout the lifetime of individual histone proteins. Although our results are consistent with lysine N6-formylation as a stable protein modification, we cannot rule out the possibility of an enzyme that removes this modification from selected conserved lysine sites in histone proteins, resulting in small changes in the population level of formyllysine that are not detectable by our current analytical method. Figure 7 summarizes our findings presented here on N6-formyllysine adducts.

Bottom Line: While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources.Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct.These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

ABSTRACT
Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6)-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6)-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6)-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6)-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4) lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4) lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

Show MeSH
Related in: MedlinePlus