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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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Sources of N6-formyllysine.The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N6-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N6-formyllysine.
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pgen-1003328-g001: Sources of N6-formyllysine.The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N6-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N6-formyllysine.

Mentions: While N6-formyllysine adducts are now well recognized as abundant protein modifications in cells, the source of these pathological adducts remains unclear. We recently showed that some portion of N6-formyllysine arises in chromatin proteins by reaction of lysine side chains with the 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA in cells (Figure 1) [9]. However, the observation of this adduct in proteins treated with the biological oxidant, peroxynitrite, suggests other sources for the formyl species [5]. Considering that formaldehyde reacts with amines to give a carbinolamine intermediate that is only one oxidation state away from a formamide functional group (Figure 1), we hypothesized that endogenous formaldehyde could serve as a source of N6-formyllysine residues in histone and other proteins. In addition to environmental and occupational sources [19]–[21], formaldehyde arises from cellular processes such as oxidative demethylation of nucleic acid and histone proteins, as well as biosynthesis of purines, thymidine, and some amino acids [20], [22], [23], making it a relatively abundant metabolite at concentrations ranging from 13 to 97 µM in human plasma [20]. To test this hypothesis and to clarify the cellular locations and quantities of N6-formyllysine relative to other major histone modifications, we developed a novel liquid chromatography-coupled electrospray tandem mass spectrometry (LC-MS/MS) method to quantify all N6-methyl-, -acetyl-, and -formyl-lysine modifications. Application of this method reveals that endogenous formaldehyde is a major source of N6-formyllysine, that this adduct is widespread among proteins in all cellular compartments, and that, in chromatin proteins, it is refractory to removal by histone deacetylases.


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)

Sources of N6-formyllysine.The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N6-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N6-formyllysine.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003328-g001: Sources of N6-formyllysine.The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N6-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N6-formyllysine.
Mentions: While N6-formyllysine adducts are now well recognized as abundant protein modifications in cells, the source of these pathological adducts remains unclear. We recently showed that some portion of N6-formyllysine arises in chromatin proteins by reaction of lysine side chains with the 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA in cells (Figure 1) [9]. However, the observation of this adduct in proteins treated with the biological oxidant, peroxynitrite, suggests other sources for the formyl species [5]. Considering that formaldehyde reacts with amines to give a carbinolamine intermediate that is only one oxidation state away from a formamide functional group (Figure 1), we hypothesized that endogenous formaldehyde could serve as a source of N6-formyllysine residues in histone and other proteins. In addition to environmental and occupational sources [19]–[21], formaldehyde arises from cellular processes such as oxidative demethylation of nucleic acid and histone proteins, as well as biosynthesis of purines, thymidine, and some amino acids [20], [22], [23], making it a relatively abundant metabolite at concentrations ranging from 13 to 97 µM in human plasma [20]. To test this hypothesis and to clarify the cellular locations and quantities of N6-formyllysine relative to other major histone modifications, we developed a novel liquid chromatography-coupled electrospray tandem mass spectrometry (LC-MS/MS) method to quantify all N6-methyl-, -acetyl-, and -formyl-lysine modifications. Application of this method reveals that endogenous formaldehyde is a major source of N6-formyllysine, that this adduct is widespread among proteins in all cellular compartments, and that, in chromatin proteins, it is refractory to removal by histone deacetylases.

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