Lysine Malonylation Is Elevated in Type 2 Diabetic Mouse Models and Enriched in Metabolic Associated Proteins

Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future. Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future. Post-translational modifications (PTMs) 1The abbreviations used are:PTMpost-translational modifications10-FTHFDH10-Formyltetrahydrofolate DehydrogenaseACAT1Acetoacetyl-CoA thiolaseALDOBfructose bisphosphate aldolase BFBP1fructose-1,6-bisphosphatase 1G6PIglucose-6-phosphate isomeraseGSTsglutathione S-transferasesHbA1chaemoglobin A1cHMGCS2Hydroxymethylglutaryl-CoA synthase, mitochondrialLDHAlactate dehydrogenase AMCDmalonyl-CoA decarboxylasePBEPeroxisomal bifunctional enzymeSAMS-adenosylmethionine. 1The abbreviations used are:PTMpost-translational modifications10-FTHFDH10-Formyltetrahydrofolate DehydrogenaseACAT1Acetoacetyl-CoA thiolaseALDOBfructose bisphosphate aldolase BFBP1fructose-1,6-bisphosphatase 1G6PIglucose-6-phosphate isomeraseGSTsglutathione S-transferasesHbA1chaemoglobin A1cHMGCS2Hydroxymethylglutaryl-CoA synthase, mitochondrialLDHAlactate dehydrogenase AMCDmalonyl-CoA decarboxylasePBEPeroxisomal bifunctional enzymeSAMS-adenosylmethionine. have been recognized as a common feature of proteins (1.Nussinov R. Tsai C.J. Xin F. Radivojac P. Allosteric post-translational modification codes.Trends Biochem. Sci. 2012; 37: 447-455Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 2.Walsh 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 (1034) Google Scholar, 3.Strahl B.D. Allis C.D. The language of covalent histone modifications.Nature. 2000; 403: 41-45Crossref PubMed Scopus (6608) Google Scholar). More than 300 types of PTMs have been identified according to the Swiss-Prot database (4.Lu C.T. Huang K.Y. Su M.G. Lee T.Y. Bretana N.A. Chang W.C. Chen Y.J. Chen Y.J. Huang H.D. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.Nucleic Acids Res. 2013; 41: D295-D305Crossref PubMed Scopus (155) Google Scholar, 5.Khoury G.A. Baliban R.C. Floudas C.A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database.Sci. Rep. 2011; 1: 90Crossref Scopus (594) Google Scholar). Most of them use small molecular compounds as group donors. For example, adenosine-triphosphate (ATP) is used in phosphorylation, S-adenosylmethionine (SAM) in methylation, and acetyl-CoA in acetylation. Lysine acylations including malonylation (6.Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (512) Google Scholar), succinylation (7.Zhang 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 (572) Google Scholar), butyrylation (8.Chen Y. Sprung R. Tang Y. Ball H. Sangras B. Kim S.C. Falck J.R. Peng J. Gu W. Zhao Y. Lysine propionylation and butyrylation are novel post-translational modifications in histones.Mol. Cell Proteomics. 2007; 6: 812-819Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar), propionylation (9.Cheng Z. Tang Y. Chen Y. Kim S. Liu H. Li S.S. Gu W. Zhao Y. Molecular characterization of propionyllysines in nonhistone proteins.Mol. Cell Proteomics. 2009; 8: 45-52Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and crotonylation (10.Tan M. Luo H. Lee S. Jin F. Yang J.S. Montellier E. Buchou T. Cheng Z. Rousseaux S. Rajagopal N. Lu Z. Ye Z. Zhu Q. Wysocka J. Ye Y. Khochbin S. Ren B. Zhao Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar) represent a group of PTMs that use intermediates of energy metabolism like malonyl-CoA, succinyl-CoA, butyryl-CoA, propionyl-CoA, and crotonyl-CoA as group donors. Among the lysine acylations, lysine malonylation was first identified in Escherichia coli (E. coli) and HeLa cells using a specific anti-Kmal (anti-malonyllysine) antibody (6.Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (512) Google Scholar). It was found in three proteins in E. coli and 17 proteins in HeLa cells. Using a novel chemical fluorescent probe, another group identified more than 300 malonylated protein candidates in HeLa cells (11.Bao X. Zhao Q. Yang T. Fung Y.M. Li X.D. A chemical probe for lysine malonylation.Angew Chem. Int. Ed. Engl. 2013; 52: 4883-4886Crossref PubMed Scopus (56) Google Scholar). Despite the rapid progress in detection technologies and tools, functional studies of lysine malonylation and its role in human diseases have been lagging behind. post-translational modifications 10-Formyltetrahydrofolate Dehydrogenase Acetoacetyl-CoA thiolase fructose bisphosphate aldolase B fructose-1,6-bisphosphatase 1 glucose-6-phosphate isomerase glutathione S-transferases haemoglobin A1c Hydroxymethylglutaryl-CoA synthase, mitochondrial lactate dehydrogenase A malonyl-CoA decarboxylase Peroxisomal bifunctional enzyme S-adenosylmethionine. post-translational modifications 10-Formyltetrahydrofolate Dehydrogenase Acetoacetyl-CoA thiolase fructose bisphosphate aldolase B fructose-1,6-bisphosphatase 1 glucose-6-phosphate isomerase glutathione S-transferases haemoglobin A1c Hydroxymethylglutaryl-CoA synthase, mitochondrial lactate dehydrogenase A malonyl-CoA decarboxylase Peroxisomal bifunctional enzyme S-adenosylmethionine. Type 2 diabetes is characterized by hyperglycemia and production of glycated proteins. For example, glycated hemoglobin A1c (HbA1c) has been clinically used as diagnostic criteria for diabetes. In addition to glycation, the role of other types of PTMs in type 2 diabetes remains to be revealed. In fact, elevated malonyl-CoA levels have been found in type 2 diabetic patients (12.Bandyopadhyay G.K. Yu J.G. Ofrecio J. Olefsky J.M. Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects.Diabetes. 2006; 55: 2277-2285Crossref PubMed Scopus (230) Google Scholar), and prediabetic rats (13.Zhao Z. Lee Y.J. Kim S.K. Kim H.J. Shim W.S. Ahn C.W. Lee H.C. Cha B.S. Ma Z.A. Rosiglitazone and fenofibrate improve insulin sensitivity of pre-diabetic OLETF rats by reducing malonyl-CoA levels in the liver and skeletal muscle.Life Sci. 2009; 84: 688-695Crossref PubMed Scopus (19) Google Scholar). And hepatic overexpression of malonyl-CoA decarboxylase (MCD) decreased malonyl-CoA and reversed insulin resistance (14.An J. Muoio D.M. Shiota M. Fujimoto Y. Cline G.W. Shulman G.I. Koves T.R. Stevens R. Millington D. Newgard C.B. Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver, and whole-animal insulin resistance.Nat. Med. 2004; 10: 268-274Crossref PubMed Scopus (365) Google Scholar). Given the use of malonyl-CoA as malonyl donor in lysine malonylation, lysine malonylation is therefore anticipated to be of functional significance in the pathogenesis of type 2 diabetes. In the present study, we observed elevated lysine malonylation in liver tissues of db/db mice after unbiased screening seven types of lysine acylations. We then detected elevated levels of lysine malonylation in liver tissues of more db/db and ob/ob mice. Using an immunoaffinity based proteomic method, we identified a total of 573 malonylated lysine sites from 268 proteins in liver tissues of wt and db/db mice. Elevation of lysine malonylation in five proteins was confirmed by immunoprecipitation coupled with Western blot analysis. Functional analysis of the malonylated proteins showed an apparent enrichment in metabolic pathways, especially those involved in the glucose and fatty acid metabolism. Our study indicates the putative association between protein lysine malonylation and type 2 diabetes. Anti-malonyllysine (PTM-901), anti-acetyllysine (PTM-105), anti-1,2-dimethyllysine (PTM-602), anti-succinyllysine (PTM-401), anti-butyryllysine (PTM-301), anti-propionyllysine (PTM-201), anti-crotonyllysine (PTM-502) antibodies, and anti-malonyllysine conjugated agarose beads were purchased from PTM Biolabs (Chicago, IL). G6PI (ab86950), LDHA (ab47010), FBP1 (ab109732), and 10-FTHFDH/ALDH1L1 (ab56777) antibodies were purchased from Abcam (Cambridge, MA). Glutathione (GSH) agarose beads were purchased from GE Healthcare Life Sciences (Uppsala, Sweden). Sequencing-grade trypsin was purchased from Promega (Madison, WI), and C18 ZipTips were purchased from Millipore (Billerica, MA). A list of other chemicals used for LC-MS/MS is available in previously published study (15.Chen X. Cui Z. Wei S. Hou J. Xie Z. Peng X. Li J. Cai T. Hang H. Yang F. Chronic high glucose induced INS-1beta cell mitochondrial dysfunction: a comparative mitochondrial proteome with SILAC.Proteomics. 2013; 13: 3030-3039Crossref PubMed Scopus (15) Google Scholar). C57 BLKS db/db mice (n = 5) and wt mice (n = 5) were provided by the Animal Center of Peking University. C57 BL/6J-ob/ob mice (n = 4) and wt mice (n = 4) were provided by the Institute of Laboratory Animal Science, CAMS (Chinese Academy of Medical Science). Mouse studies were approved by the Animal Experimentation Ethics Committee (Institute of Biophysics, CAS) and the National Health and Medical Research Council of China Guidelines on Animal Experimentation. Mice were sacrificed by cervical dislocation. Tissues were homogenized in 1 ml lysis buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS). The homogenized tissues were centrifuged at 12,000 rpm and the supernatant containing the tissue lysates was kept for later steps. Western blot and Coomassie blue staining analysis were carried out as described previously (16.Jiang P. Huang Z. Zhao H. Wei T. Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease.Biochem. Biophys. Res. Commun. 2013; 433: 408-414Crossref PubMed Scopus (16) Google Scholar). In vitro malonylation was performed as previously described (17.Wagner G.R. Payne R.M. Widespread and enzyme-independent Nepsilon-acetylation and Nepsilon-succinylation of proteins in the chemical conditions of the mitochondrial matrix.J. Biol. Chem. 2013; 288: 29036-29045Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Briefly, FLAG-tagged ALDOB was overexpressed in 293T cells and purified using ANTI-FLAG M2 Affinity Gel (A2220, Sigma-Aldrich, St. Louis, MO) as per instruction by the manufacturer. Purified ALDOB proteins were incubated for 12 h at 37 °C in buffer containing 1 mm malonyl-CoA, 50 mm Tris-Cl, pH 8.0, and 150 mm NaCl. Enzymatic activity of ALDOB was determined spectrophotometrically by measuring the decrease of NADH at 340 nm in a coupled assay with triosephosphate isomerase (TPI) and glycerophosphate dehydrogenase (GDH) (18.Santamaria R. Esposito G. Vitagliano L. Race V. Paglionico I. Zancan L. Zagari A. Salvatore F. Functional and molecular modeling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase.Biochem. J. 2000; 350: 823-828Crossref PubMed Scopus (17) Google Scholar). Assays were performed in triplicates at 30 °C in buffer A (100 mm Tris-Cl, pH 7.5, and 1 mm EDTA) following addition of fructose 1,6-bisphosphate. For in-gel digestion, protein bands around 95 KDa, 55 KDa, 34 KDa, and 26 KDa were excised from the SDS-PAGE gels, cut into small pieces and destained using 50 mm triethyl ammonium bicarbonate (TEAB) in 40% acetonitrile. 100% acetonitrile was used to dehydrate the gel. 5 mm Tris (2-carboxyethyl) phosphine (TCEP) was used to reduce the proteins. 10 mm methyl methanethiosulfonate (MMTS) was used to alkylate the proteins. Digestion was carried out using sequencing-grade modified trypsin (Promega) with a substrate ratio of 1:50 (w/w) overnight at 37 °C. Peptides were extracted with 60% acetonitrile and 5% formic acid (FA). Resulting peptides were dried and prepared for immunoprecipitation. For in-solution digestion, liver tissue lysates were precipitated with acetone and resolved in 8 m urea with 50 mm Tris-HCl, pH 8.0. The samples were reduced and alkylated as done for in-gel digestion. The tryptic peptides obtained from in-gel digestion or in-solution digestion were resolubilized in NETN buffer (50 mm Tris-Cl, pH 8.0, 0.5% Nonidet P-40, 100 mm NaCl, and 1 mm EDTA). Anti-malonyllysine antibody-conjugated agarose beads were added and incubated at 4 °C for 12 h with gentle shaking. The beads were washed two times with NETN buffer, once with ETN buffer (50 mm Tris-Cl, pH 8.0, 100 mm NaCl and 1 mm EDTA), and once with purified water. The bound peptides were eluted by 0.1% trifluoroacetic acid (TFA) and dried using the SpeedVac. The dried peptides were re-dissolved in 0.1% FA and desalted using C18 ZipTips. LC-MS/MS analyses were performed on a LTQ-Orbitrap mass spectrometer (Thermo, San Jose, CA) equipped with an Eksigent nanoLC system (Eksigent Technologies, LLC, Dublin, CA). The peptide mixtures were loaded onto 20-mm ReproSil-Pur C18-AQ (Dr. Maisch GmbH, Ammerbuch) trapping columns (packed in-house, i.d., 150 μm; resin, 5 μm) and eluted into 150 mm ReproSil-Pur C18-AQ (Dr. Maisch GmbH, Ammerbuch) analytical columns (packed in-house, i.d., 75 μm; resin, 3 μm) for MS analysis. Elution was achieved with a 0–35% gradient buffer B (Buffer A, 0.1% formic acid and 5% acetonitrile; Buffer B, 0.1% formic acid and 95% acetonitrile) over 90 min using a data-dependent method. All MS/MS spectra were collected using normalized collision energy (setting, 35%), an isolation window of 3 m/z, and one micro-scan. The acquired MS/MS spectra were searched using the Mascot (v2.3.2) search engine in the Proteome Discoverer™ Software (v1.3, Thermo Fisher Scientific, Bremen, Germany) against the mouse Uniprot database (version 2.3, 50,807 sequences) using a target-decoy database searching strategy (19.Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2834) Google Scholar). The following search criteria were employed: three missed cleavages were allowed; mass tolerance for parent ions was set to 20 ppm; mass tolerance for fragment ions was set to 0.8 Da; minimum peptide length was set at 6; Cys (+45.9877 Da, methanethiosulfonate, MMTS) was set as a fixed modification; Met (+15.9949 Da, oxidation) and Lys (+86.0004 Da, malonylation) were considered as variable modifications. The global false discovery rate (FDR) for both peptides and proteins was set to 0.01. The relative quantitation of proteins between two samples (wt versus db/db mice) was achieved by extracted ion chromatograms (XICs) of peptides with a Mascot score of ≥25 that ranked first. The resulting peptides were extracted using the Precursor Ion Area Detector feature in the Proteome Discoverer program (version 1.3), using a mass tolerance of 3 ppm. Only proteins with a minimum of two quantifiable peptides were included in our final data set. The peptides did not match any proteins present in the Uniprot database, and peptides with no XICs in both wt and db/db mice were excluded from the final data set. The malonylated proteins were classified based on the PANTHER (Protein Analysis through Evolutionary Relationships) system. The DAVID 6.7 was employed for enrichment analysis of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The default mouse proteome was used as the background list. The significance of the obtained enrichments was evaluated by using the Benjamini-Hochberg corrected p value. The identified proteins with lysine malonylation were searched against the STRING database (V 9.1) for protein–protein interactions. A network of protein–protein interactions was generated, which was then visualized using the Cytoscape program (v3.0.2). This interaction network was further analyzed for densely connected regions using the program “Molecular Complex Detection” (MCODE). The db/db mice, which has a homozygous point mutation in the db gene encoding for the leptin receptor, is an classical type 2 diabetic mouse model (20.Coleman D.L. Diabetes-obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar, 21.Hummel K.P. Dickie M.M. Coleman D.L. Diabetes, a new mutation in the mouse.Science. 1966; 153: 1127-1128Crossref PubMed Scopus (747) Google Scholar). To investigate the role of protein lysine acylations in type 2 diabetes, seven types of lysine acylations were investigated by Western blot in liver tissue lysates of wt and db/db mice. A marked elevation of lysine malonylation was observed in db/db mice relative to wild type littermates (Fig. 1A), while little elevation of lysine acetylation, 1, 2-methylation, succinylation, butyrylation, propionylation, and crotonylation was detected (Fig. 1B–1G). Equal loading was verified by β-actin Western blot and Coomassie blue staining (Fig. 1H, I). Furthermore, we detected lysine malonylation in another four wt and db/db mice and observed a significant increase in lysine malonylation in liver tissues of all db/db mice (Fig. 2A). To investigate whether lysine malonylation levels are elevated beyond wt levels in other type 2 diabetic animal models as well, we tested lysine malonylation levels in ob/ob mice. Consistent with the observations in db/db mice, an elevated levels of lysine malonylation was detected in liver tissues of all ob/ob mice relative to wt mice (Fig. 2B). Together, these findings demonstrated that lysine malonylation was elevated in liver tissues of db/db and ob/ob type 2 diabetic animal models.Fig. 2Detection of lysine malonylation in liver of four db/db and ob/ob mice. Western blot analysis of liver tissue lysates from four db/db (db) A, and ob/ob (ob) B, mice and their counterpart wt mice by anti-malonyllysine antibody. Equal loading was verified using β-actin Western blot and Coomassie blue staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To validate the increase of protein lysine malonylation and identify the malonylated lysine sites and proteins, liver lysates from three wt or db/db mice were combined and proteins were separated by SDS-PAGE. Four gel bands with the highest levels of lysine malonylation were cut and trypsin-digested (Fig. 3A). For analysis of each band, a total of 50 gels were cut and combined. The digested peptides were immunoprecipitated with anti-malonyllysine antibody-conjugated agarose beads and then loaded onto LC-MS/MS. One representative MS/MS spectrum of the identified malonylated peptides is shown in Fig. 3B. There was a neutral loss of carbon dioxide (CO2, 44 Da), which was used for confirming the presence of lysine malonylation. The LC-MS/MS results showed that the number of malonylated proteins and peptides identified for each of the four bands was higher in db/db mice relative to wt (Fig. 3C). Detailed information on all malonylated proteins and peptides identified is summarized in supplemental Table S1. Together, our results indicated that proteins from the livers of db/db mice were more intensively malonylated than those in wt, which was consistent with the results obtained from Western blot analysis. Having successfully tested the ability of our LC-MS/MS method to detect malonylation, we used in-solution tryptic digestion to widen the scope of our analysis and identify lysine malonylation in a larger number of peptides (Fig. 4A). Liver lysates were combined from either three wt or three db/db mice, then trypsin-digested and immunoprecipitated using anti-malonyllysine-conjugated agarose beads. After immunoprecipitation, LC-MS/MS, and a search against the mouse Uniprot database, we identified a total of 268 Uniprot-reviewed proteins, containing 573 malonylated lysine sites (supplemental Table S2). There were 246 lysine malonylation sites that overlapped between wt and db/db mice (Fig. 4B); and 169 malonylated proteins that overlapped between the two groups (Fig. 4C). We extracted ion chromatograms and calculated the area under the peak associated with malonylated peptides, which indirectly reflects the quantity of each malonylated peptide. For the 246 overlapping peptides, the average under-the-curve area of extracted ion chromatograms for each malonylated peptides in db/db was larger than that in the wt mice (supplemental Fig. S1). Together, these results suggested greater malonylation in db/db livers relative to wt, and were consistent with the results obtained by Western blot and in-gel identification. We compared the 268 malonylated proteins in the mouse liver with two previously acquired malonylation data sets in the HeLa cell line by manually searching for protein homologs. Our data contained 13 malonylated proteins out of 17 proteins identified by Zhao et al. and 54 malonylated protein candidates out of the 375 proteins identified by Li et al. (supplemental Table S3) (6.Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (512) Google Scholar, 11.Bao X. Zhao Q. Yang T. Fung Y.M. Li X.D. A chemical probe for lysine malonylation.Angew Chem. Int. Ed. Engl. 2013; 52: 4883-4886Crossref PubMed Scopus (56) Google Scholar). Thus, the overlap observed between these data sets confirmed the validity of our experimental procedures and results. We then compared our data with a large-scale succinylation data set, and 135 out of 433 Uniprot-reviewed proteins were shown to overlap between the two data sets (supplemental Table S3) (22.Park J. Chen Y. Tishkoff D.X. Peng C. Tan M. Dai L. Xie Z. Zhang Y. Zwaans B.M. Skinner M.E. Lombard D.B. Zhao Y. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.Mol. Cell. 2013; 50: 919-930Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). These results suggested that malonylation and succinylation might occur at identical proteins. Our data also revealed that some proteins were highly malonylated. For example, 29 malonylated peptides (19 unique malonylated lysine sites) were identified in the one-carbon metabolic enzyme 10-FTHFDH. The proteins with more than five malonylated peptides are summarized in supplemental Table S4. To evaluate the biological relevance of lysine malonylation, the PANTHER classification system was used to sort the malonylated proteins according to their associated biological processes (23.Mi H. Muruganujan A. Casagrande J.T. Thomas P.D. Large-scale gene function analysis with the PANTHER classification system.Nat. Protoc. 2013; 8: 1551-1566Crossref PubMed Scopus (1690) Google Scholar). The results of this analysis showed that 194 out of 268 malonylated proteins (72%) were classified as involved in a metabolic process (Fig. 4D and supplemental Table S5). The analysis of Gene Ontology (GO) terms by the Functional Annotation Tool of DAVID also revealed that most of the enriched GO terms were involved in catabolic or metabolic processes (supplemental Table S5) (24.Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25477) Google Scholar). To better understand the function of lysine malonylation, the malonylated proteins were subjected to a protein–protein interaction (PPI) network analysis using the STRING database (25.Franceschini A. Szklarczyk D. Frankild S. Kuhn M. Simonovic M. Roth A. Lin J. Minguez P. Bork P. von Mering C. Jensen L.J. STRING v9.1: protein–protein interaction networks, with increased coverage and integration.Nucleic Acids Res. 2013; 41: D808-D815Crossref PubMed Scopus (3297) Google Scholar). A PPI network containing 239 nodes and 1517 edges were constructed. To characterize protein complexes among the malonylated proteins, the PPI networks was analyzed for highly connected nodes by MCODE (26.Bader G.D. Hogue C.W. An automated method for finding molecular complexes in large protein interaction networks.BMC Bioinformatics. 2003; 4: 2Crossref PubMed Scopus (3837) Google Scholar). Five highly connected clusters have been identified including the glutathione metabolic, oxoacid metabolic, organic acid metabolic, glucose catabolic, and translation cluster (Fig. 4E). The PPI network analysis indicated that lysine malonylation occurs in various metabolic complexes. To further characterize the role of lysine malonylation, KEGG pathway analysis was performed for the malonylated proteins. The results showed that malonylated proteins were enriched in the glucose, fatty acid, and amino acid metabolic pathways (supplemental Table S5 and Fig. 5A). Disorders of glucose and fatty acid metabolism are two major features of type 2 diabetes. The majority of enzymes involved in glucose and fatty acid metabolism were identified to be malonylated in livers of db/db mice (Fig. 5B and supplemental Table S2). Indeed, many of the enzymes identified showed elevated levels of lysine malonylation (supplemental Table S6). Dysfunction of glucose metabolic enzymes, including glucokinase (27.Glaser B. Kesavan P. Heyman M. Davis E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. Familial hyperinsulinism caused by an activating glucokinase mutation.N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (519) Google Scholar, 28.Froguel P. Vaxillaire M. Sun F. Velho G. Zouali H. Butel M.O. Lesage S. Vionnet N. Clement K. Fougerousse F. Tanizawa Y. 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