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.
Highlights
The positively charged lysine residue plays an important role in protein folding and functions
These results reveal that lysine propionylation and butyrylation are novel lysine modifications that can be catalyzed by acetyltransferases
Initial Identification of Lysine-propionylated and -butyrylated Peptides in Histone H4 Protein—We hypothesized that propionyl-CoA could be used by acetyltransferases for lysine propionylation
Summary
In-gel Digestion—Protein in-gel digestion, peptide extraction, and peptide cleaning using a -C18 ZipTip were carried out as reported previously [18]. HPLC/MS/MS Analysis—HPLC/MS/MS analysis for mapping propionylation and butyrylation sites in histone H4, p53, p300, and CBP were carried out by nano-HPLC/LTQ mass spectrometry as described previously [6]. The LTQ mass spectrometer was operated in the data-dependant mode acquiring fragmentation spectra of the 10 strongest ions. Protein Sequence Database Search and Manual Verification—All MS/MS spectra were searched against the National Center for Biotechnology Information non-redundant (NCBI-nr) protein sequence database (updated November 28, 2006 with 4,125,643 entries) specifying lysine modifications using the MASCOT database search engine (version 2.1). All lysine-propionylated or -butyrylated peptides identified with MASCOT score Ͼ20.0 were manually examined with the rules described previously [19], and all lysine propionylation or butyrylation sites had to be identified by consecutive b- or y-ions so that the possibility that propionylation (ϩ56 Da) or butyrylation (ϩ70 Da) occurred on adjacent residues was eliminated. The reaction mixture was subjected to electrophoresis by SDS-PAGE followed by either autoradiography or Coomassie Blue staining
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