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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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Analysis of lysine demethylation as a source of N6-formyllysine.Methyl groups in N6-methyllysine species in TK6 cells were labeled using L-methionine-([13C,2H3]-methyl) and N6-formyllysine and N6-methyllysine species were quantified by LC-MS/MS as described in Materials and Methods. Panels A and B: N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 m/z and 8 m/z, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N6-[13C, 2H]-formyllysine (177 m/z→114 m/z transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N6-formyllysine.
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pgen-1003328-g005: Analysis of lysine demethylation as a source of N6-formyllysine.Methyl groups in N6-methyllysine species in TK6 cells were labeled using L-methionine-([13C,2H3]-methyl) and N6-formyllysine and N6-methyllysine species were quantified by LC-MS/MS as described in Materials and Methods. Panels A and B: N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 m/z and 8 m/z, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N6-[13C, 2H]-formyllysine (177 m/z→114 m/z transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N6-formyllysine.

Mentions: The enzymatic demethylation of N6-methyllysine modifications represents another possible source of N6-formyllysine in histone proteins, given both the carbinolamine intermediate known to form during the process of lysine demethylation and the ultimate release of the methyl group as formaldehyde [23]. Adventitious oxidation of the carbinolamine intermediate or secondary reaction of the released formaldehyde could result in the formation of N6-formyllysine locally. To test these hypotheses, TK6 cells were grown in customized RPMI medium containing L-methionine with a [13C,2H3]-methyl group for 20 days to label all methyl groups in N6-methyllysine species, and histone proteins were extracted for analysis every 2 days. If N6-formyllysine is a product of disrupted lysine demethylation in histones and is formed via oxidation of the carbinolamine intermediate known to form during the process of lysine demethylation [23], or by reaction of lysine with the formaldehyde released at the last step of successful lysine demethylation [23], then one would expect to see an increase of 2 mass units corresponding to formation of N6-[13C, 2H]-formyllysine (m/z 177→114 transition). In order to increase the signal-to-noise ratio for N6-[13C,2H]-formyllysine, N6-formyllysine was HPLC-pre-purified in all samples before LC-MS/MS analysis. Figure 5 depicts an example of the analysis using the day 6 sample. As shown in Figure 5, N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (i.e.,[13C,2H3]-methyl). In contrast to methyllysines, the level of N6-[13C,2H]-formyllysine did not increase beyond the natural isotope abundance level of ∼0.7% for the [M+2] ion of N6-formyllysine (Figure 5C and Figure S2). Note that the HPLC gradient was changed here to fully resolve a contaminant signal from the TK6 cells (identified as the [M+1] ion of citrulline) that otherwise co-eluted with N6-formyllysine and produced an m/z value similar to the [M+2] isotopomer of N6-formyllysine.


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)

Analysis of lysine demethylation as a source of N6-formyllysine.Methyl groups in N6-methyllysine species in TK6 cells were labeled using L-methionine-([13C,2H3]-methyl) and N6-formyllysine and N6-methyllysine species were quantified by LC-MS/MS as described in Materials and Methods. Panels A and B: N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 m/z and 8 m/z, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N6-[13C, 2H]-formyllysine (177 m/z→114 m/z transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N6-formyllysine.
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Related In: Results  -  Collection

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

pgen-1003328-g005: Analysis of lysine demethylation as a source of N6-formyllysine.Methyl groups in N6-methyllysine species in TK6 cells were labeled using L-methionine-([13C,2H3]-methyl) and N6-formyllysine and N6-methyllysine species were quantified by LC-MS/MS as described in Materials and Methods. Panels A and B: N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 m/z and 8 m/z, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N6-[13C, 2H]-formyllysine (177 m/z→114 m/z transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N6-formyllysine.
Mentions: The enzymatic demethylation of N6-methyllysine modifications represents another possible source of N6-formyllysine in histone proteins, given both the carbinolamine intermediate known to form during the process of lysine demethylation and the ultimate release of the methyl group as formaldehyde [23]. Adventitious oxidation of the carbinolamine intermediate or secondary reaction of the released formaldehyde could result in the formation of N6-formyllysine locally. To test these hypotheses, TK6 cells were grown in customized RPMI medium containing L-methionine with a [13C,2H3]-methyl group for 20 days to label all methyl groups in N6-methyllysine species, and histone proteins were extracted for analysis every 2 days. If N6-formyllysine is a product of disrupted lysine demethylation in histones and is formed via oxidation of the carbinolamine intermediate known to form during the process of lysine demethylation [23], or by reaction of lysine with the formaldehyde released at the last step of successful lysine demethylation [23], then one would expect to see an increase of 2 mass units corresponding to formation of N6-[13C, 2H]-formyllysine (m/z 177→114 transition). In order to increase the signal-to-noise ratio for N6-[13C,2H]-formyllysine, N6-formyllysine was HPLC-pre-purified in all samples before LC-MS/MS analysis. Figure 5 depicts an example of the analysis using the day 6 sample. As shown in Figure 5, N6-mono-methyllysine and N6-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (i.e.,[13C,2H3]-methyl). In contrast to methyllysines, the level of N6-[13C,2H]-formyllysine did not increase beyond the natural isotope abundance level of ∼0.7% for the [M+2] ion of N6-formyllysine (Figure 5C and Figure S2). Note that the HPLC gradient was changed here to fully resolve a contaminant signal from the TK6 cells (identified as the [M+1] ion of citrulline) that otherwise co-eluted with N6-formyllysine and produced an m/z value similar to the [M+2] isotopomer of N6-formyllysine.

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