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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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Effect of lysine deacetylases on N6-formyllysine.(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, 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). (B) Treatment of peptide substrates containing N6-acetyllysine or N6-formyllysine with the class III histone deacetylase, SIRT1.
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pgen-1003328-g006: Effect of lysine deacetylases on N6-formyllysine.(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, 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). (B) Treatment of peptide substrates containing N6-acetyllysine or N6-formyllysine with the class III histone deacetylase, SIRT1.

Mentions: The chemical similarity of N6-formyllysine and N6-acetyllysine suggested that the former might be subject to removal by lysine deacetylases that recognize and remove N6-acetyllysine from histone and other proteins [16], [27]–[30]. Lysine deacetylases fall into several classes, including classes I and II that share a common hydrolytic mechanism and are all inhibited by suberoylanilidehydroxamic acid (SAHA), and the class III enzymes (i.e., sirtuins) that are NAD+-dependent deacetylases [31], [32]. In order to assess the activity of lysine deacetylases with N6-formyllysine substrates, TK6 cells were treated with SAHA and the levels of N6-acetyllysine and N6-formyllysine were quantified. The results shown in Figure 6A reveal that, while SAHA caused a 3-fold increase in the level of N6-acetyllysine (4 to 14 per 103 lysines), lysine formylation was not affected. To assess sirtuin activity against N6-formyllysine, we performed in vitro reactions of SIRT1 with a consensus peptide (GGAKRHR) containing N6-formyllysine or N6-acetyllysine, and the quantities of the modified and unmodified peptides were analyzed by LC-MS/MS. As shown in Figure 6B, SIRT1 removed the acetyl modification completely to generate the unmodified peptide, while only ∼10% (±4%) of N6-formyllysine was removed.


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)

Effect of lysine deacetylases on N6-formyllysine.(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, 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). (B) Treatment of peptide substrates containing N6-acetyllysine or N6-formyllysine with the class III histone deacetylase, SIRT1.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003328-g006: Effect of lysine deacetylases on N6-formyllysine.(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, 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). (B) Treatment of peptide substrates containing N6-acetyllysine or N6-formyllysine with the class III histone deacetylase, SIRT1.
Mentions: The chemical similarity of N6-formyllysine and N6-acetyllysine suggested that the former might be subject to removal by lysine deacetylases that recognize and remove N6-acetyllysine from histone and other proteins [16], [27]–[30]. Lysine deacetylases fall into several classes, including classes I and II that share a common hydrolytic mechanism and are all inhibited by suberoylanilidehydroxamic acid (SAHA), and the class III enzymes (i.e., sirtuins) that are NAD+-dependent deacetylases [31], [32]. In order to assess the activity of lysine deacetylases with N6-formyllysine substrates, TK6 cells were treated with SAHA and the levels of N6-acetyllysine and N6-formyllysine were quantified. The results shown in Figure 6A reveal that, while SAHA caused a 3-fold increase in the level of N6-acetyllysine (4 to 14 per 103 lysines), lysine formylation was not affected. To assess sirtuin activity against N6-formyllysine, we performed in vitro reactions of SIRT1 with a consensus peptide (GGAKRHR) containing N6-formyllysine or N6-acetyllysine, and the quantities of the modified and unmodified peptides were analyzed by LC-MS/MS. As shown in Figure 6B, SIRT1 removed the acetyl modification completely to generate the unmodified peptide, while only ∼10% (±4%) of N6-formyllysine was removed.

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