Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extramitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.
Bibliographical noteFunding Information:
Our work was supported by National Institutes of Health (NIH) award R01GM101171 (D.B.L.), the National Technology Center for Networks and Pathways (U54GM103520; Y.Z.), the Ellison Medical Foundation (AG-NS-0583-09; D.B.L.), and the National Science and Technology Major Project of the Ministry of Science and Technology of China (2012ZX09301001-007; M.T.). J.P. and D.X.T. were supported in part by a National Institute on Aging training grant (T32AG000114). Work in the Lombard laboratory was also supported by DP3DK094292 and P30AG013283 and by pilot awards from the Michigan Diabetes Research and Training Center (P60DK020572), the Michigan Metabolomics and Obesity Center (P30DK089503), and the Claude D. Pepper Older American’s Independence Center (P30AG024824). The authors acknowledge S. Pletcher for help with statistical analysis; C. Burant and R. Harris, and members of the Lombard and Zhao labs for helpful discussions; and C. Fry and Y. Li (Cell Signaling Technology) for SIRT5 antibodies. Y.Z. is a shareholder and a member of the scientific advisory board of PTM BioLabs. J.P. and D.X.T. carried out respiration and enzymatic studies. Y.C., M.T., L.D., and Y.Z. performed the HPLC fractionation, affinity enrichment, HPLC-MS/MS, and bioinformatic data analysis. C.P., Z.X., J.P., D.T., and B.M.M.Z. generated MEF cell lines and performed immunoblot analysis. J.P., B.M.M.Z., and M.E.S. performed mouse husbandry. D.B.L. and Y.Z. conceived the project and supervised the experiments. D.B.L., Y.C., J.P., and Y.Z. wrote the manuscript.