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. 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. 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 (1Walsh C.T. Garneau-Tsodikova S. Gatto Jr., G.J. Protein posttranslational modifications: the chemistry of proteome diversifications.Angew. Chem. Int. Ed. Engl. 2005; 44: 7342-7372Crossref PubMed Scopus (1055) Google Scholar, 2Walsh C.T. Posttranslational Modifications of Proteins: Expanding Nature's Inventory. Roberts and Co. Publishers, Greenwood Village, CO2006Google Scholar, 3Schweppe R.E. Haydon C.E. Lewis T.S. Resing K.A. Ahn N.G. The characterization of protein post-translational modifications by mass spectrometry.Acc. Chem. Res. 2003; 36: 453-461Crossref PubMed Scopus (75) Google Scholar). 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 (1Walsh C.T. Garneau-Tsodikova S. Gatto Jr., G.J. Protein posttranslational modifications: the chemistry of proteome diversifications.Angew. Chem. Int. Ed. Engl. 2005; 44: 7342-7372Crossref PubMed Scopus (1055) Google Scholar). 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 (4Allfrey V.G. Faulkner R. Mirsky A.E. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis.Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1787) Google Scholar), the modification was later identified in non-histone proteins, such as p53 (5Gu W. Shi X.L. Roeder R.G. Synergistic activation of transcription by CBP and p53.Nature. 1997; 387: 819-823Crossref PubMed Scopus (523) Google Scholar). A recent proteomics screening showed that acetyllysine is abundant and present in substrates that are affiliated with multiple organelles and have diverse functions (6Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). 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 (6Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). The modification plays important roles in diverse cellular processes, such as apoptosis, metabolism, transcription, and the stress response (7Blander G. Guarente L. The Sir2 family of protein deacetylases.Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1313) Google Scholar, 8Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. The biochemistry of sirtuins.Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (587) Google Scholar, 9Roth S.Y. Denu J.M. Allis C.D. Histone acetyltransferases.Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar, 10Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation?.EMBO J. 2000; 19: 1176-1179Crossref PubMed Scopus (1010) Google Scholar). In addition to their roles in fundamental biology, lysine acetylation and its regulatory enzymes (acetyltransferases and deacetylases) are intimately linked to aging (11Haigis M.C. Guarente L.P. Mammalian sirtuins—emerging roles in physiology, aging, and calorie restriction.Genes Dev. 2006; 20: 2913-2921Crossref PubMed Scopus (1093) Google Scholar) and several major diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases (12McKinsey T.A. Olson E.N. Cardiac histone acetylation—therapeutic opportunities abound.Trends Genet. 2004; 20: 206-213Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Yang X.J. The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases.Nucleic Acids Res. 2004; 32: 959-976Crossref PubMed Scopus (425) Google Scholar, 14Hake S.B. Xiao A. Allis C.D. Linking the epigenetic ‘language’ of covalent histone modifications to cancer.Br. J. Cancer. 2004; 90: 761-769Crossref PubMed Scopus (312) Google Scholar). 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 (15King M.T. Reiss P.D. Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography.Anal. Biochem. 1985; 146: 173-179Crossref PubMed Scopus (79) Google Scholar). Finally it appears, from structural studies on some HATs 1The abbreviations used are: HAT, histone acetyltransferase; CBP, CREB (cAMP-response element-binding protein)-binding protein; PCAF, p300/CBP-associated factor. (such as Hat1), that the enzyme has ample space within the cofactor binding pocket to accept propionyl-CoA without steric interference (16Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Structure of the yeast histone acetyltransferase Hat1: insights into substrate specificity and implications for the Gcn5-related N-acetyltransferase superfamily.Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 501-507Crossref PubMed Scopus (18) Google Scholar). 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 (17Nelson D.L. Cox M.M. Lehninger Principles of Biochemistry. W. H. Freeman and Co., New York2005: 631-652Google Scholar), 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. Protein in-gel digestion, peptide extraction, and peptide cleaning using a μ-C18 ZipTip were carried out as reported previously (18Zhao Y. Zhang W. Kho Y. Zhao Y. Proteomic analysis of integral plasma membrane proteins.Anal. Chem. 2004; 76: 1817-1823Crossref PubMed Scopus (230) Google Scholar). 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 (6Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). Briefly each tryptic digest was dissolved in 10 μl of HPLC buffer A (0.1% formic acid in water (v/v)), and 2 μl were injected into an Agilent HPLC system (Agilent, Palo Alto, CA). Peptides were separated on a home-made capillary HPLC column (50-mm length × 75-μm inner diameter, 4-μm particle size, 90-Å pore diameter) with Jupiter C12 resin (Phenomenex, St. Torrance, CA) and directly electrosprayed into the mass spectrometer using a nanospray source. The LTQ mass spectrometer was operated in the data-dependant mode acquiring fragmentation spectra of the 10 strongest ions. 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). A low cutoff peptide score of 20.0 was selected to maximize the identification of lysine-modified peptides. For each Mascot search, the peptide mass error was set to ±4 Da, fragment ion mass error was set to ±0.6 Da, and six missing cleavages were allowed. All lysine-propionylated or -butyrylated peptides identified with MASCOT score >20.0 were manually examined with the rules described previously (19Chen Y. Kwon S.W. Kim S.C. Zhao Y. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra.J. Proteome Res. 2005; 4: 998-1005Crossref PubMed Scopus (161) Google Scholar), and all lysine propionylation or butyrylation sites had to be identified by consecutive b- or y-ions so that the possibility that propionylation (+56 Da) or butyrylation (+70 Da) occurred on adjacent residues was eliminated. The synthesis procedure can be found in the supplemental information. In vitro propionylation and butyrylation assays were carried out essentially as described previously (20Gu W. Roeder R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar) with some modifications. Tagged human FLAG-p300, hemagglutinin-CBP, FLAG-MOF, and FLAG-PCAF proteins from transfected 293 cells and tagged human GST-Tip60 and GST-p53 expressed in bacteria were purified to homogeneity under stringent conditions (500 mm NaCl + 1% Triton X-100). Ten-microliter reactions contained 50 mm Tris, pH 7.9, 10% glycerol, 1 mm DTT, 10 mm sodium butyrate, 1 μl of [14C]acyl-CoA (55 mCi/mmol; acetyl-CoA from Amersham Biosciences and propionyl-CoA and butyryl-CoA from American Radiolabeled Chemicals, Inc.). Two and a half micrograms of substrates (human core histones and recombinant human histone H4 (Upstate Biotechnology, Lake Placid, NY) or GST-p53) and 20–100 ng of the enzyme protein, as indicated, were incubated at 30 °C for 1 h. The reaction mixture was then subjected to electrophoresis by SDS-PAGE followed by either autoradiography or Coomassie Blue staining. We hypothesized that propionyl-CoA could be used by acetyltransferases for lysine propionylation. In addition, we further assumed that some propionylated, tryptic peptides could be affinity-purified by anti-acetyllysine antibody due to the close structural similarity between the acetyllysine residue and the propionyllysine residue (Fig. 1). To identify lysine-propionylated peptides, we searched the MS/MS datasets of affinity-enriched acetyllysine-containing tryptic peptides acquired in nano-HPLC/LTQ mass spectrometry on lysine acetylation proteomics was previously (6Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1231) Google the protein sequence database search, the lysine was as or The database search and manual of peptide to the identification of lysine-propionylated histone H4 peptides for MS/MS The identification of lysine-propionylated peptides to for the lysine-butyrylated peptides. The datasets were searched with the lysine as or The analysis identified two histone H4 peptides with lysine butyrylation sites for MS/MS of lysine-propionylated and -butyrylated peptides identified in vivo and the of modification sites identified in proteins in of of vitro in a Two of MS/MS spectra are to the identification of lysine-propionylated and lysine-butyrylated peptides (Fig. 2 and In the peptide identification by protein sequence database search, the in to the identification of lysine-propionylated 1 that in to the identification of lysine-propionylated 2 A of the spectra revealed that were a of fragment in spectra with that could not be to the fragment from 1 and the of lines of evidence suggest that the spectra were from peptide with mass difference of were in the peptides have the molecular and the peptides the in could be for by two lysine-butyrylated and and and an lysine-propionylated peptide and could be identified from the in The nature of an identified peptide can be by MS/MS of their corresponding synthetic peptides, a for of peptide identification and To the of the propionylated and butyrylated peptides in MS/MS of synthetic peptides with the and modification corresponding to Peptides and in were and To the in vivo fragmentation by the peptide mixture in synthetic peptides were at a of MS/MS analysis of the peptide mixture in a fragmentation (Fig. that with the in vivo in the identification of one peptide with lysine propionylation and two peptides with lysine butyrylation. to Peptides 2 and identified from the in vivo in two corresponding synthetic peptides were and The fragmentation of a peptide mixture at a with the in vivo in the identification of two lysine-propionylated peptides. we identified in vivo lysine propionylation at and as well as in vivo lysine butyrylation at and of histone H4 The and of histone H4 are known to be is the of lysine methylation Lysine acetylation at the H4 lysine residues is with high structure, and DNA and histone Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Histone H4 lysine acetylation the Biol. 2006; PubMed Scopus Google Scholar). of the acetyllysine residues of histone a to a enzyme T. Chen G. the histone acetylation for a human Full Text Full Text PDF PubMed Scopus Google Scholar). Although biological functions of lysine propionylation and butyrylation in histones unknown, it is to that propionyllysine or be involved in the of a distinct set of to the structure of and histone H4 can be propionylated and butyrylated in we whether core histones can be propionylated and butyrylated in vitro by acetyltransferases using either or acetyltransferases were CBP, p300, and CBP and p300 are known acetyltransferases for and of histone The core histones were incubated with one of the CoAs and the acetyltransferase of The protein mixture was then by SDS-PAGE and by CBP and p300 showed significant in modifications on histone H4 (Fig. the other significant propionylation or butyrylation were for the other acetyltransferases, and To evidence of an in vitro modification reaction at lysine residues, we used analysis to the lysine-modified residues in histone and were found to be propionylated and butyrylated by CBP these establish that histone H4 can be lysine-propionylated and -butyrylated directly by CBP and p300 in vitro. To whether acetyltransferases can catalyze lysine propionylation and butyrylation reactions in non-histone proteins, we in vitro propionylation and butyrylation reactions in of p53, its and its biological functions (5Gu W. Shi X.L. Roeder R.G. Synergistic activation of transcription by CBP and p53.Nature. 1997; 387: 819-823Crossref PubMed Scopus (523) Google Scholar, V. A. of in 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). lysine residues in p53 can be some of which are known to be modified by (5Gu W. Shi X.L. Roeder R.G. Synergistic activation of transcription by CBP and p53.Nature. 1997; 387: 819-823Crossref PubMed Scopus (523) Google Scholar, Y. J. Zhang W. W. acetylation of p53 the between and Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. M. A. DNA p53 a Dev. 1998; PubMed Scopus Google Scholar). Given the that are the acetyltransferases for p53 and that have for lysine propionylation and butyrylation in histones, we whether the HATs could catalyze similar reactions in we the in vitro reactions for p53 using the procedure described only two of the acetyltransferases, CBP and p300, could carry out propionylation and butyrylation reactions at p53 at a significant reaction under our conditions (Fig. Interestingly p300 showed than CBP for In the two enzymes had histones. CBP and p300 are acetyltransferases that can catalyze To whether the proteins can carry out autopropionylation and autobutyrylation we the modification sites at p300 and CBP by mass lysine propionylation sites and lysine butyrylation sites were in p300, lysine propionylation sites and lysine butyrylation sites were in CBP and supplemental of propionylated and butyrylated peptides in non-histone proteins, p53, CBP, and p300, the possibility that the two modifications are not to histones. In we report the identification of in vivo lysine propionylation and lysine butyrylation in histones. The modifications were by synthetic peptides and in vitro acetyltransferases, we showed that two previously known acetyltransferases, CBP and p300, could catalyze lysine modification reactions using either propionyl-CoA or in histones and p53, at a significant reaction in vitro. Lysine is one of the two major ribosomally coded amino acid residues with a charged side chain at an important role in protein folding and functions. We not will be the functional of the two modifications. Nevertheless the regulatory functions of known lysine modification and significant structural changes induced by lysine propionylation and it is anticipated that the two modifications are likely to have biological functions in the fashion as other lysine modifications. are known to be modified by an of post-translational modifications, including methylation, acetylation, ubiquitination, and W. Y. Allis C.D. Histone and Biol. 2003; PubMed Scopus Google Scholar). A of post-translational modifications in histones, the the functions of the proteins in and T. Allis C.D. the histone 2001; PubMed Scopus Google Scholar). modifications of histones have been by biochemistry T. Allis C.D. the histone 2001; PubMed Scopus Google Scholar) and mass spectrometry S.B. M. J. J. Allis C.D. in post-translational modifications in histones and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.T. characterization of human histones in the family by mass Proteome Res. 2006; PubMed Scopus Google Scholar, Zhang S. Allis C.D. of histone and posttranslational by ion mass spectrometry Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Nevertheless lysine propionylation and butyrylation at histones have not been reported results suggest that the complexity of histone modifications remains to be can the of and amino is only from acid and amino acid butyryl-CoA is a metabolic the of as well as a substrate for acid The of the short-chain CoAs on and cellular conditions (15King M.T. Reiss P.D. Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography.Anal. Biochem. 1985; 146: 173-179Crossref PubMed Scopus (79) Google Scholar). the of the modifications on the of the short-chain CoAs, directly or it be to that lysine propionylation and butyrylation regulate cellular metabolic in response to cellular a then the for the to to and regulatory The and of regulatory enzymes for propionylation and butyrylation be by other For some the of the CoA binding pocket the binding to the short-chain it is possible that the binding be modulated by other regulatory molecules, such as of or proteins. Finally identification of lysine propionylation and lysine butyrylation further the possible of novel enzymes for the modifications than the known of lysine propionylation and butyrylation are the enzymes for the of lysine propionylation and Given the close structural some of the HATs and histone be to catalyze the the of a of lysine modifications: acetylation, and are the biological regulated by lysine propionylation and The described here is the of future into the fundamental of the novel lysine modifications. We Zhang for for with