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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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Addition of [13C,2H2]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N6-formyllysine.(A) LC-MS/MS analysis showing signals for the three isotopomeric N6-formyllysine species, as described in Materials and Methods. (B) Plot of N6-formyllysine levels as a function of exposure to [13C,2H2]-formaldehyde. Data represent mean ± SD for N = 3.
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pgen-1003328-g004: Addition of [13C,2H2]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N6-formyllysine.(A) LC-MS/MS analysis showing signals for the three isotopomeric N6-formyllysine species, as described in Materials and Methods. (B) Plot of N6-formyllysine levels as a function of exposure to [13C,2H2]-formaldehyde. Data represent mean ± SD for N = 3.

Mentions: The relatively high endogenous levels of N6-formyllysine in histone and other proteins (Table 1 and Table 2) raised the question of the contribution of exogenous formaldehyde exposures to the total load of N6-formyllysine in the cells. To address this issue, we exposed TK6 cells to [13C,2H2]-labeled formaldehyde, which led to the formation of N6-[13C,2H]-formyllysine that is 2 mass units heavier than the endogenous adducts (Figure 4A). Following extraction of the histone proteins from formaldehyde-treated TK6 cells (2 h, 37°C), both endogenous and exogenous N6-formyllysine were quantified by monitoring the transitions m/z 175→112 and m/z 177→114, respectively (Figure 4A), with a third transition (m/z 179→116) for the 4,4,5,5-[2H]-N6-formyllysine internal standard. As shown in Figure 4B, levels of endogenous (unlabeled) N6-formyllysine remained constant at all concentrations of [13C,2H2]-formaldehyde, while N6-[13C,2H]-formyllysine increased as a function of the concentration of labeled formaldehyde.


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)

Addition of [13C,2H2]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N6-formyllysine.(A) LC-MS/MS analysis showing signals for the three isotopomeric N6-formyllysine species, as described in Materials and Methods. (B) Plot of N6-formyllysine levels as a function of exposure to [13C,2H2]-formaldehyde. Data represent mean ± SD for N = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003328-g004: Addition of [13C,2H2]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N6-formyllysine.(A) LC-MS/MS analysis showing signals for the three isotopomeric N6-formyllysine species, as described in Materials and Methods. (B) Plot of N6-formyllysine levels as a function of exposure to [13C,2H2]-formaldehyde. Data represent mean ± SD for N = 3.
Mentions: The relatively high endogenous levels of N6-formyllysine in histone and other proteins (Table 1 and Table 2) raised the question of the contribution of exogenous formaldehyde exposures to the total load of N6-formyllysine in the cells. To address this issue, we exposed TK6 cells to [13C,2H2]-labeled formaldehyde, which led to the formation of N6-[13C,2H]-formyllysine that is 2 mass units heavier than the endogenous adducts (Figure 4A). Following extraction of the histone proteins from formaldehyde-treated TK6 cells (2 h, 37°C), both endogenous and exogenous N6-formyllysine were quantified by monitoring the transitions m/z 175→112 and m/z 177→114, respectively (Figure 4A), with a third transition (m/z 179→116) for the 4,4,5,5-[2H]-N6-formyllysine internal standard. As shown in Figure 4B, levels of endogenous (unlabeled) N6-formyllysine remained constant at all concentrations of [13C,2H2]-formaldehyde, while N6-[13C,2H]-formyllysine increased as a function of the concentration of labeled formaldehyde.

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