Chao Peng

Protein post-translational modifications (PTMs) at the lysine residue, such as lysine methylation, acetylation, and ubiquitination, are diverse, abundant, and dynamic. They play a key role in the regulation of diverse cellular physiology. Here we report discovery of a new type of lysine PTM, lysine malonylation (Kmal). Kmal was initially detected by mass spectrometry and protein sequence-database searching. The modification was comprehensively validated by Western blot, tandem MS, and high-performance liquid chromatography of synthetic peptides, isotopic labeling, and identification of multiple Kmal substrate proteins. Kmal is a dynamic and evolutionarily conserved PTM observed in mammalian cells and bacterial cells. In addition, we demonstrate that Sirt5, a member of the class III lysine deacetylases, can catalyze lysine demalonylation and lysine desuccinylation reactions both in vitro and in vivo. This result suggests the possibility of nondeacetylation activity of other class III lysine deacetylases, especially those without obvious acetylation protein substrates. Our results therefore reveal a new type of PTM pathway and identify the first enzyme that can regulate lysine malonylation and lysine succinylation status. Protein post-translational modifications (PTMs) at the lysine residue, such as lysine methylation, acetylation, and ubiquitination, are diverse, abundant, and dynamic. They play a key role in the regulation of diverse cellular physiology. Here we report discovery of a new type of lysine PTM, lysine malonylation (Kmal). Kmal was initially detected by mass spectrometry and protein sequence-database searching. The modification was comprehensively validated by Western blot, tandem MS, and high-performance liquid chromatography of synthetic peptides, isotopic labeling, and identification of multiple Kmal substrate proteins. Kmal is a dynamic and evolutionarily conserved PTM observed in mammalian cells and bacterial cells. In addition, we demonstrate that Sirt5, a member of the class III lysine deacetylases, can catalyze lysine demalonylation and lysine desuccinylation reactions both in vitro and in vivo. This result suggests the possibility of nondeacetylation activity of other class III lysine deacetylases, especially those without obvious acetylation protein substrates. Our results therefore reveal a new type of PTM pathway and identify the first enzyme that can regulate lysine malonylation and lysine succinylation status. Cellular function and physiology are largely determined by the inventory of all proteins in a cell, its proteome. The collection and characterization of the proteome is critical to understanding cellular mechanisms and diseases. Proteomes in eukaryotic cells consist of over a million molecular species of proteins, easily orders of magnitude more complex than the corresponding genomes (1Witze E.S. Old W.M. Resing K.A. Ahn N.G. Mapping protein post-translational modifications with mass spectrometry.Nat. Methods. 2007; 4: 798-806Crossref PubMed Scopus (611) Google Scholar, 2Walsh 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). There are two major mechanisms for expanding the coding capacity of the human genome: mRNA splicing and protein post-translational modifications (PTMs) 1The abbreviations used are:PTMProtein post-translational modificationKATlysine acetyltransferaseHDACHistone lysine deacetylaseMS/MStandem MSHPLChigh performance liquid chromatography. . Protein post-translational modification lysine acetyltransferase Histone lysine deacetylase tandem MS high performance liquid chromatography. PTMs (more than 300 types) are complex and fundamental mechanisms of cellular regulation, and have been associated with almost all known cellular pathways and disease processes (1Witze E.S. Old W.M. Resing K.A. Ahn N.G. Mapping protein post-translational modifications with mass spectrometry.Nat. Methods. 2007; 4: 798-806Crossref PubMed Scopus (611) Google Scholar, 2Walsh 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). As an example, protein phosphorylation, the most well-studied PTM, is present in more than one third of human proteins, the phosphorylation status of which can potentially be regulated by ∼500 human protein kinases and ∼150 phosphatases (3Johnson S.A. Hunter T. Kinomics: methods for deciphering the kinome.Nat. Methods. 2005; 2: 17-25Crossref PubMed Scopus (349) Google Scholar, 4Denu J.M. Dixon J.E. Protein tyrosine phosphatases: mechanisms of catalysis and regulation.Curr. Opin. Chem. Biol. 1998; 2: 633-641Crossref PubMed Scopus (336) Google Scholar). The modification mainly occurs at several amino acid residues: serine, threonine, tyrosine, and histidine. Protein phosphorylation makes its substrate residues more acidic, hydrophilic, and induces a charge change from +1 charge to –1 (at physiological pH), which in turn modulates the structure and functions of substrate proteins. The high complexity of PTMs is also reflected by diverse modifications at ε-amine group of lysine residue, including methylation, acetylation, and ubiquitination. These lysine PTMs have been shown to play an important role in cellular regulations (5Guarente L. Sirtuins in aging and disease.Cold Spring Harb. Symp Quant Biol. 2007; 72: 483-488Crossref PubMed Scopus (292) Google Scholar, 6Martin C. Zhang Y. The diverse functions of histone lysine methylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1615) Google Scholar). Recently, we identified a new type of PTM at lysine residues, lysine succinylation (7Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (588) Google Scholar). Like phosporylation, lysine succinylation also induces a change of two negative charges in lysine residue, from a +1 charge to a –1 charge at physiological pH. This PTM is dynamic, abundant, and evolutionarily conserved from bacteria to mammalian cells. We anticipate that there will be more than a thousand lysine succinylated substrates in mammalian cells. Here we report the identification and verification of a new type of PTM, lysine malonylation (Fig. 1). We extensively confirmed this new modification using a variety of chemical and biochemical methods. We showed that Sirt5 can catalyze lysine demalonylation and desuccinylation reactions, in peptide and protein substrates, both in vivo and in vitro. Taken together, we conclusively establish lysine malonylation as a new type of evolutionarily conserved PTM pathway and Sirt5 is a regulatory enzyme for lysine malonylation and lysine succinylation. Peptides were synthesized through custom synthesis using racemic or enantiomeric protected amino acid residues. The synthesis of the protected amino acid residues was described in detail in supplementary Methods, including Fmoc-Lys(mono-tert-butyl malonate)-OH, Fmoc-Lys(succinyl)-OH. Ni-NTA agarose beads were purchased from Qiagen (Valencia, CA); modified sequencing-grade trypsin from Promega (Madison, WI); C18 ZipTips from Millipore Corporation (Billerica, MA); water and acetonitrile from Burdick and Jackson (Meskegon, MI); protein A beads from GE Healthcare; isotopic [1, 2, 3-13C] sodium malonate was purchased from Cambridge Isotope Laboratories (Andover, MA). The recombinant HDACs except Sirt5 were purchased from BPS Bioscience (San Diego, CA). Wt Sirt5 and its inactive mutant proteins were expressed and purified in house. Experimental procedure for expression and purification of Sirt5 were included in the supplementary Methods. Expression plasmids for Sirt5 wt and its inactive mutant (H158Y) were constructed by Wei Gu (Columbia University) and Eric Verdin (UCSF), respectively. Other chemicals were obtained from the following suppliers: Sigma-Aldrich: trifluoroacetic acid (99%), formic acid (>98%), NH4HCO3 (>99%), trichloroacetic acid (6.1 N), iodoacetamide, dithothreitol, bovine serum albumin (BSA), sodium dodecyl sulfate, Tris-Cl, MgCl2 (ACS grade), NaCl (ACS grade), KCl (ACS grade), β-nicotinamide adenine dinucleotide hydrate (NAD+), nicotinamide (ACS grade), trichostatin A (TSA), sirtinol (ACS grade); Fisher: NaHCO3 (ACS grade), NaOH (ACS grade), CH3CN (HPLC grade), HCl solution (37.3%), glycine (ACS grade), MeOH (ACS grade), acetone (ACS grade), DMEM medium and hydrogen peroxide (ACS grade). Chemical synthesis of Fmoc-Lys (mono-tert-butyl malonate)-OH and fluorescent compounds for assaying lysine demalonylation and lysine desuccinylation activities were included in the supplementary Methods. The peptides were synthesized by commercial custom synthesis using Fmoc-Lys (mono-tert-butyl malonate)-OH. Generation of anti-Kmal antibody was described in supplementary Methods. The specificity of the sequence-specific antibody was demonstrated by dot-blot assay (Fig. 2A). To carry out Western blot, 40 μg of each cell lysate was resolved in gradient SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membrane; to carry out dot-blot assay, a gradient amount of peptide was spotted to the nitrocellulose (NC) membrane. Then the membrane was blocked in TBST buffer (1x TBS containing 0.05% Tween 20) with 5% BSA and probed with appropriate primary antibodies and secondary antibodies coupled to horseradish peroxidase. The competition experiments were performed by incubating the primary antibodies with a synthetic peptide bearing either an unmodified lysine or a modified lysine residue. Signals were revealed by ECL Western blot detection system. HeLa cells were treated with isotopic sodium [1, 2, 3-13C] malonate (20 mm in DMEM medium, pH 7.5) for 24 h. The cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed with SDS sample buffer. The protein lysate was used either for Western blot analysis or for identifying Kmal peptides by immunoprecipitation using anti-Kmal antibody and mass spectrometry. The protein lysate of interest was precipitated by conventional trichloroacetic acid method. The protein precipitate was digested by trypsin using a procedure we described previously (8Kim 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 protein precipitate was suspended in 100 mm NH4HCO3 (pH 8.0) and dissolved by sonication. Trypsin was added to the mixture at an enzyme-to-substrate ratio of 1:50 (w/w) and tryptic digestion was carried out at 37 °C for 16 h. The tryptic peptides were reduced by 5 mm dithothreitol and alkylated by 15 mm iodoacetamide. The mixture was digested again by adding an additional trypsin (1:100 w/w) and incubated at 37 °C for 3 h. The Kmal peptides were enriched from proteolytic digests of protein cell lysate using a procedure described previously (8Kim 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, the tryptic peptides obtained above were resolubilized in NETN buffer (50 mm Tris-Cl, pH 8.0, 0.5% Nonidet P-40, 100 mm NaCl, 1 mm EDTA). Insoluble particles were removed by centrifugation at 50,000 × g for 10 min. Affinity purification was carried out by incubating the peptides with 10 μl of anti-Kmal antibody protein A immobilized agarose beads at room temperature for 4 h with gentle shaking. The beads were washed three times with NETN buffer and twice with ETN buffer (50 mm Tris-Cl, pH 8.0, 100 mm NaCl, 1 mm EDTA). The bound peptides were eluted from the beads by washing three times with 0.1% TFA. The isolated Kmal peptides were in The peptide sample was dissolved in buffer A formic acid in with C18 ZipTips and an C18 × to an were with a from 5 to buffer formic acid in at a of The eluted peptides were and a mass using a MS were in the with at The most were isolated in the and to with a of The for the was the was 2, and the was at and was and are for were for for in and for in for was at The was by for were at and for were at were by the were either Protein human protein or protein by iodoacetamide, and lysine malonylation were as of three were the results with above and 1 were to have high and The in vivo the synthetic and mixture for each Kmal peptide were to washing was carried out to sample carry over each sample were with at and were at a of at in The of synthetic peptides were that the of the in vivo peptide were a from its synthetic The reactions were performed in a of μl in a Briefly, one μl of or solution and HDACs were added to the buffer mm Tris-Cl, mm NaCl, mm 1 mm 0.1% pH 8.0) and incubated at 37 °C for h. assaying activities of μg protein of interest and 1 μl of the solution were added to the buffer. The was then by adding μl of trypsin solution mm mm NaCl, mm 1 mm pH The was and incubated at 37 °C for h or appropriate The was a with at and at We identified lysine succinylation as a new type of is an abundant, evolutionarily and dynamic PTM (7Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (588) Google Scholar). we carried out of substrates by using an and protein we detected the of a peptide from a To for Kmal peptides, we an anti-Kmal which was shown to have high specificity lysine antibody detected its peptide and peptide bearing a peptide bearing a unmodified succinylated lysine or a peptide bearing an unmodified (Fig. 2A). To Kmal in we performed Western blot analysis using anti-Kmal antibody the cell from and HeLa cells. were detected that be by its a peptide with a Kmal residue, the corresponding unmodified succinylated or peptide bearing a succinylated lysine (Fig. multiple can be detected with the antibody These results that the Kmal PTM is present in both mammalian and bacterial cells. To identify Kmal substrates and malonylation in proteins, we used a previously described for the proteomics of lysine acetylation (8Kim 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). In this we used anti-Kmal antibody to Kmal peptides from a tryptic of HeLa lysate and The Kmal peptides were by The were by protein to identify the Kmal A verification was used to peptide identification using a procedure we previously described Y. S.C. Zhao Y. for verification of peptides identified by protein with tandem mass 2005; 4: PubMed Scopus Google Scholar). the are included in supplementary This to the identification of peptides from proteins of HeLa cell and three peptides from three proteins of that each a mass of to of the identified peptides are in HeLa of all the Kmal peptides identified by MS from HeLa the results were the results were in a new Taken together, the results of Western blot competition assay, and proteomics using the of Kmal in both mammalian and bacterial cells. peptides will the and times in the analysis of corresponding synthetic peptides with the PTM of interest is the to that a peptide a new To the Kmal peptides identified in we Kmal peptides for chemical The peptides were using the in vivo peptide and its synthetic As an example, the of a peptide isolated from HeLa cell the as that of a synthetic peptide with a group and a mixture of the two peptides (Fig. In addition, the in vivo peptide with the synthetic peptide in (Fig. These two of confirmed that the mass of in the is by lysine we also confirmed lysine malonylation in other peptides by and analysis using corresponding synthetic peptides and and can be and which can be used in turn by cells for lysine acetylation and succinylation reactions, (7Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (588) Google Scholar, R. histone acetylation in of PubMed Scopus Google Scholar). We that malonate also be used by cells in a as was previously and A in PubMed Scopus Google Scholar, acid in of 2: PubMed Scopus Google Scholar). To this we first HeLa cells with mm malonate for 24 h. malonylation status was then in HeLa lysate by Western blot analysis with anti-Kmal Our result showed that lysine malonylation was by with sodium malonate (Fig. To malonate can be to a high such as that is in turn used by cells for lysine we HeLa cells with mm isotopic sodium [1, 2, for 24 h. We protein from lysate that was then digested with The tryptic peptides were to using the anti-Kmal and enriched peptides were by and peptide for peptide The Kmal peptides can easily be identified by an additional over the lysine This to the identification of that were with [1, 2, (Fig. and supplementary These results that malonate can be to an which be in turn used by cells as a for lysine malonylation Peptides bearing a PTM have a mass in that is the PTM example, a bearing a phosphorylation in either a or to the to a in J. of in an mass 1998; PubMed Scopus Google Scholar, S.A. detection and of at the by mass PubMed Scopus Google Scholar). This is mainly by of the in either the or mass the group is also easily in a mass to a with a of Z. identification and of J. PubMed Scopus Google Scholar, L. J.M. Mapping of modification using for and post-translational Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). we of Kmal peptides, we that almost all the peptide in of synthetic peptides bearing a Kmal have a with a mass of which to the of We that this is by a that occurs of acid C. Scholar). this the lysine first the through a which to a group This mass can be used for the identification of peptides bearing a Kmal The status of cellular lysine acetylation is regulated by two of with lysine and lysine all of the HDACs have deacetylase we that HDACs be in lysine To this we a assay to the lysine demalonylation activity of all the In this assay, a synthetic substrate was incubated with each of the A with lysine demalonylation activity the group from the modified lysine residue, a proteolytic digestion for a tryptic substrates will an this assay, we the lysine demalonylation activity of all HDACs and This assay showed that Sirt5 lysine demalonylation and that its inactive mutant have demalonylation activity (Fig. of lysine demalonylation activity of Sirt5 to activities lysine a PTM we previously (7Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (588) Google Scholar). Sirt5 is again the that lysine desuccinylation activity in this assay (Fig. In to lysine demalonylation and lysine desuccinylation Sirt5 showed activity lysine in this assay, which is from high activities of To the activities of Sirt5 in we carried out a lysine demalonylation using synthetic peptides bearing Kmal residues We used mass spectrometry to the demalonylation of this As an example, the molecular of a synthetic from is (Fig. with Sirt5, an additional with a mass of a of for a was detected in the of corresponding to peptide that a In the peptide was detected in a and in a using a Sirt5 inactive mutant or the peptide bearing an lysine The demalonylation and can be by nicotinamide class III trichostatin A an for class and sirtinol of and human (Fig. This result that Sirt5 can catalyze lysine demalonylation in a Kmal peptide in vitro and is the of this we also that Sirt5 can catalyze a lysine demalonylation in vitro for other lysine substrates To the desuccinylation activity of Sirt5 in we also carried out lysine desuccinylation reactions using three synthetic peptides bearing residues As an example, the molecular of a synthetic from is (Fig. with Sirt5, an additional with a mass of a of for a was detected in the of corresponding to the peptide that a In the peptide was detected in a and in a using a Sirt5 Like lysine demalonylation desuccinylation activity of Sirt5 and can be by or we also showed that Sirt5 can catalyze lysine desuccinylation reactions in vitro for other two lysine succinylated substrates for a lysine for the corresponding peptides To the lysine demalonylation and lysine desuccinylation activities of Sirt5 in proteins, we resolved protein lysate in SDS-PAGE and transferred the proteins from the to the membrane. We then incubated the recombinant Sirt5 with the membrane (Fig. Western blot showed that lysine malonylation and lysine succinylation are in multiple protein This result that Sirt5 can catalyze the lysine demalonylation and lysine succinylation in vitro. To the result of the in vitro we the lysine malonylation and lysine succinylation status in in which Sirt5 was or by These experiments demonstrated that lysine malonylation or lysine succinylation were in to Sirt5 expression the protein lysate from from wt Sirt5 and Sirt5 we showed that Sirt5 the lysine malonylation and lysine lysine acetylation (Fig. Taken together, we demonstrated that Sirt5 can catalyze lysine demalonylation and lysine desuccinylation both in vitro and in vivo. with a at physiological an important role in protein functions and lysine and lysine acetylation have been extensively in the important in regulation and In this we the identification of lysine malonylation in both mammalian and bacterial cells. This modification was validated by Western blot, competition assay, proteomics and synthetic peptides in with malonylation induces more than both lysine acetylation and methylation, and therefore is to have the substrate proteins. The class III of three of and Sirt5, are known to be in is the substrate identified to Sirt5 was shown to this protein and its activity T. L. 1 and the Full Text Full Text PDF PubMed Scopus Google Scholar). was that Sirt5 be a major deacetylase in for two obvious was detected in or Cheng J. R. J. Yang Y. Chen Y. M. S. Verdin lysine Cell Biol. 2007; PubMed Scopus Google Scholar). In protein that the protein is an important human Sirt5 was to have deacetylase activity in vitro J.M. Verdin The human is an Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we showed demalonylation and desuccinylation activities of The activities were initially identified using using recombinant proteins. The in vitro demalonylation and desuccinylation activities were confirmed using peptide and protein substrates. The activities were confirmed in vivo by Western blot using experiments and from both and We showed that Sirt5 activities to lysine malonylation and lysine succinylation than lysine acetylation, both in vitro and in vivo. HDACs were in the of which have obvious protein substrates. Identification of Sirt5 as a demalonylation and desuccinylation enzyme suggests a possibility of nondeacetylation activities of other especially those that have known substrates. the other Sirt5 is the first enzyme we identified that demalonylation and desuccinylation this result the possibility of the of such activities in other proteins. is an important in the pathways of cellular is known to be synthesized by three major the of by A the of by and of can be in cells through acid a by acid and through to by In to an important was also shown to have regulatory of and in the of J. 2011; PubMed Scopus Google Scholar). As an important and regulatory of cellular physiology. example, an of is by activity A PubMed Scopus Google Scholar). acid reduced mass and more They are also protected and by high L. mutant are protected and by PubMed Scopus Google Scholar). These an and Jr., for the of Opin. Google Scholar). The critical of in that lysine malonylation of cellular regulations in to the dynamic of cellular and have is a in and of and amino is that cells either one of the two PTM pathways succinylation and lysine the to its cellular is also that regulatory are the two PTM as in the of lysine acetylation and lysine Identification of lysine malonylation and Sirt5 as the first regulatory enzyme for lysine malonylation is to be the first for the for other regulatory and for characterization of functions of lysine lysine malonylation a new in the cellular regulation, such as in diverse with