Molecular anatomy of post-translational modifications that regulate cellular processes and disease progression stands as one of the major goals of postgenomics biological research. To date, more than 200 post-translational modifications have been described, providing an efficient way to diversify the primary structure of a protein and possibly its functions (1
). The remarkable complexity of these molecular networks is exemplified by modifications at the side chain of lysine, one of the 15 ribosomally coded amino acid residues known to be modified (1
). The electron-rich and nucleophilic nature of the lysine side chain makes it suitable for undergoing covalent post-translational modification reactions with diverse substrates. The residue can be potentially modulated by several post-translational modifications including methylation, acetylation, biotinylation, ubiquitination, and sumoylation, which have pivotal roles in cell physiology and pathology.
Lysine acetylation is an abundant, reversible, and highly regulated post-translational modification. Although initially discovered in histones (4
), the modification was later identified in non-histone proteins, such as p53 (5
). A recent proteomics screening showed that acetyllysine is abundant and present in substrates that are affiliated with multiple organelles and have diverse functions (6
). Interestingly the modification is enriched in mitochondrial proteins and metabolic enzymes, implying its roles in fine tuning the functions of the organelle and energy metabolism (6
). The modification plays important roles in diverse cellular processes, such as apoptosis, metabolism, transcription, and the stress response (7
). In addition to their roles in fundamental biology, lysine acetylation and its regulatory enzymes (acetyltransferases and deacetylases) are intimately linked to aging (11
) and several major diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases (12
Acetyl-CoA, a member of the high energy CoA compounds, is the substrate used by acetyltransferases to catalyze the lysine acetylation reaction. It remains unknown, however, whether cells can use other short-chain CoAs, such as propionyl- and butyryl-CoA (which are structurally close to acetyl-CoA), to carry out similar post-translational modifications at lysine. Nevertheless several lines of evidence suggest such a possibility. First, like acetyl-CoA, propionyl-CoA and butyryl-CoA are high energy molecules, making it thermodynamically feasible to carry out a reaction with a lysine side chain. Second, propionyl-CoA and butyryl-CoA are structurally similar to acetyl-CoA with a difference of only one or two CH2
. Third, propionyl-CoA and butyryl-CoA are present at high concentrations in cells. In the case of starved mouse liver, the concentrations of the two CoAs are only 1–3 times less than acetyl-CoA (15
). Finally it appears, from structural studies on some HATs1
(such as Hat1), that the enzyme has ample space within the cofactor binding pocket to accept propionyl-CoA without steric interference (16
). Despite such evidence, the short-chain CoAs with the exception of acetyl-CoA have not been described as a substrate for protein modification.
Here we report the identification and validation of two novel post-translational protein modifications, propionylation and butyrylation at lysine residues, by a proteomics study. The unbiased global screening involved exhaustive peptide identification by nano-HPLC/MS/MS analysis, protein sequence database search, and manual verification. The resulting propionylated and butyrylated peptides were verified by MS/MS of their corresponding synthetic peptides. Using in vitro
labeling with isotopic propionyl-CoA and butyryl-CoA as well as mass spectrometry, we identified two acetyltransferases, p300 and CBP, that could perform robust lysine modifications at histones in vitro
. Furthermore we demonstrated that p300 and CBP could carry out autopropionylation and autobutyrylation at lysine residues in a fashion similar to autoacetylation. Taken together, these results reveal that lysine propionylation and butyrylation are novel lysine modifications that can be catalyzed by acetyltransferases. Given the unique roles of propionyl-CoA and butyryl-CoA in energy metabolism (17
), their distinct structure, and significant structural changes induced by the modifications, it is anticipated that lysine propionylation and butyrylation will have important but likely distinct functions in the regulation of biological processes. The identification of lysine-propionylated and lysine-butyrylated substrates described here provides an entry point for future functional studies of the two modifications in cellular physiology and pathology.