Lysine propionylation and butyrylation are novel post-translational modifications in histones

Abstract

The positively charged lysine residue plays an important role in protein folding and functions. Neutralization of the charge often has a profound impact on the substrate proteins. Accordingly all the known post-translational modifications at lysine have pivotal roles in cell physiology and pathology. Here we report the discovery of two novel, in vivo lysine modifications in histones, lysine propionylation and butyrylation. We confirmed, by in vitro labeling and peptide mapping by mass spectrometry, that two previously known acetyltransferases, p300 and CREB-binding protein, could catalyze lysine propionylation and lysine butyrylation in histones. Finally p300 and CREB-binding protein could carry out autopropionylation and autobutyrylation in vitro. Taken together, our results conclusively establish that lysine propionylation and lysine butyrylation are novel post-translational modifications. Given the unique roles of propionyl-CoA and butyryl-CoA in energy metabolism and the significant structural changes induced by the modifications, the two modifications are likely to have important but distinct functions in the regulation of biological processes.

FIG. 1

A, structures of three short-chain CoAs, the acetyl-CoA, propionyl-CoA, and butyryl-CoA, as well as the three modified lysines, acetyllysine, propionyllysine, and butyryllysine. B, an illustration of novel lysine propionylation and butyrylation sites in histone H4 (unmarked labels, lysine acetylation and methylation sites identified previously; circled labels, novel, in vivo lysine modification sites identified in this study; boxed labels, additional novel, in vitro lysine modification sites identified in this study). The known sites of lysine acetylation and methylation were obtained from Millipore/Upstate. Ac, acetyl; Me, methyl; Buty, butyryl; Prop, propionyl.

FIG. 2. Identification and verification of lysine-propionylated and lysine-butyrylated histone H4 peptides

A, tandem mass spectrum of a tryptic peptide ion from a peptide mixture that was affinity-enriched with an anti-acetyllysine antibody from tryptic peptides of HeLa nuclear extracts. The spectrum was used to identify one lysine-propionylated and two lysine-butyrylated peptides, Peptides 1, 12, and 13 (Table I) from histone H4. The fragmentation ions for each peptide are available in the supplemental information. B, tandem mass spectrum of a peptide mixture from the three synthetic peptides corresponding to the sequences identified in A, showing similar ion intensity. C, MS/MS of a tryptic peptide ion led to the identification of two modified peptides, Peptides 2 and 3 (Table I) from histone H4. D, tandem mass spectrum of a peptide mixture from the two synthetic peptides corresponding to the sequences identified in C, showing similar ion intensity.

FIG. 3. In vitro propionylation and butyrylation of core histones and p53 by acetyltransferases

A, in vitro propionylation and butyrylation of core histones. The core histone proteins were incubated with the purified acetyltransferase in the presence of either [¹⁴C]propionyl-CoA or [¹⁴C]butyryl-CoA as indicated. The reaction products were resolved by SDS-PAGE and visualized by autoradiography (top panel). The amounts of the core histone substrates and the acetyltransferases are shown in the middle and bottom panels, respectively, by Coomassie Blue staining. B, in vitro propionylation and butyrylation of p53. The GST-p53 recombinant protein was incubated with the purified acetyltransferase in the presence of [¹⁴C]acyl-CoA as indicated. The reaction mixtures were resolved by SDS-PAGE and visualized by autoradiography (upper panel). The amount of the GST-p53 substrate is shown in the lower panel stained by Coomassie Blue.