SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target

Summary Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD + -dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5 −/− mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5 −/− animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5 −/− compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.