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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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Formaldehyde is a source of N6-formyllysine.Formation of N6-formyllysine in (A) in vitro reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).
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pgen-1003328-g003: Formaldehyde is a source of N6-formyllysine.Formation of N6-formyllysine in (A) in vitro reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).

Mentions: One alternative to 3′-formylphosphate residues as a source of N6-formyllysine is oxidation of the carbinolamine intermediate in the reaction of formaldehyde with side chain amine of lysine (Figure 1; N6-(hydroxymethyl)-lysine). To test this hypothesis, we performed a series of experiments, starting with an in vitro reaction of L-lysine with different concentrations of formaldehyde and quantification of N6-formyllysine. As shown in Figure 3A, there was a concentration-dependent formation of N6-formyllysine in reactions with formaldehyde, presumably as a result of oxidation of the carbinolamine adduct by the background of reactive oxygen species generated by trace metals and dissolved oxygen in the solution. The oxygen dependence of formaldehyde-induced N6-formyllysine was verified by bubbling 100% oxygen (4 h) into the solution of 1 mM lysine and 10 mM formaldehyde, which caused a 2.2 (±0.4)-fold increase in the level of N6-formyllysine after 12 h of incubation at 37°C. The dose-response relationship for formaldehyde-induced N6-formyllysine formation was also observed in histone proteins extracted from TK6 cells exposed to formaldehyde for 2 h at 37°C (Figure 3B), with 10 mM formaldehyde producing roughly the same fold-change of N6-formyllysine in both in vitro and cellular studies.


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)

Formaldehyde is a source of N6-formyllysine.Formation of N6-formyllysine in (A) in vitro reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003328-g003: Formaldehyde is a source of N6-formyllysine.Formation of N6-formyllysine in (A) in vitro reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in Materials and Methods. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).
Mentions: One alternative to 3′-formylphosphate residues as a source of N6-formyllysine is oxidation of the carbinolamine intermediate in the reaction of formaldehyde with side chain amine of lysine (Figure 1; N6-(hydroxymethyl)-lysine). To test this hypothesis, we performed a series of experiments, starting with an in vitro reaction of L-lysine with different concentrations of formaldehyde and quantification of N6-formyllysine. As shown in Figure 3A, there was a concentration-dependent formation of N6-formyllysine in reactions with formaldehyde, presumably as a result of oxidation of the carbinolamine adduct by the background of reactive oxygen species generated by trace metals and dissolved oxygen in the solution. The oxygen dependence of formaldehyde-induced N6-formyllysine was verified by bubbling 100% oxygen (4 h) into the solution of 1 mM lysine and 10 mM formaldehyde, which caused a 2.2 (±0.4)-fold increase in the level of N6-formyllysine after 12 h of incubation at 37°C. The dose-response relationship for formaldehyde-induced N6-formyllysine formation was also observed in histone proteins extracted from TK6 cells exposed to formaldehyde for 2 h at 37°C (Figure 3B), with 10 mM formaldehyde producing roughly the same fold-change of N6-formyllysine in both in vitro and cellular studies.

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