SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks

Abstract

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

Figure 1. Generation of succinyl-lysine-specific antibodies and characterization of succinylation distribution in mouse tissues, cultured cells and subcellular compartments

(A) The structure of lysine succinylation and the catalytic reaction of desuccinylation by SIRT5. The succinyl group on the lysine residue is indicated in red. (B) Succinyl-lysine antibodies specifically detected succinyl-BSA, but not acetyl-BSA or butyryl- or propionyl-BSA. (C) Western blot using succinyl-lysine-specific antibodies was performed to assess succinylation and acetylation levels in mouse liver, skeletal muscle, primary cultured mouse hepatocytes and MEFs derived from wild-type (WT) or Sirt5^−/− mice (See also Figure S1A). Equal amount of proteins were loaded into each lane. Loading controls, α-tubulin and mitochondrial protein HSP70, were also examined. Western blot for SIRT5 confirmed the absence of SIRT5 protein in Sirt5^−/− mouse tissues or cells. (D) Protein succinylation and acetylation in whole cell lysates, cytoplasmic or mitochondrial fractions derived from WT or Sirt5^−/− mouse livers. Western blot for cytoplasmic protein α-tubulin and mitochondrial protein voltage dependent anion channel (VDAC) confirmed the purity of subcellular fractionation. Western blot for SIRT5 showed the presence of SIRT5 in both cytoplasm and mitochondria of WT livers, but not in Sirt5^−/− livers.

Figure 2. Enrichment and identification of liver mitochondrial lysine succinylome by label-free quantitation

(A) Liver mitochondria were isolated from five individual WT and Sirt5^−/− mice. Mitochondrial protein from each of the 10 samples was digested separately with trypsin, desalted, and 150 fmol of a heavy isotope-labeled succinyl-lysine peptide standard was added. Succinyl-lysine-containing peptides were immunoprecipitated and analyzed in duplicate by LC-MS/MS. Precursor ion intensity chromatograms were integrated using MS1 Filtering in Skyline and for label-free quantitation. Venn diagrams of lysine succinylated proteins and peptides identified in WT and Sirt5^−/− are shown in Figure S2. (B) Overlap of the number of succinylated peptides and proteins identified with the number that were targeted by SIRT5 (> twofold increase and p <0.01). For peptide information and quantitation of individual sites see Dataset S1 and S2. (C) Scatter plot of the SuK peptides quantitated by MS1 filtering. Dashed lines indicate the fold change of abundance in KO samples in comparison to WT samples. Peptides with a significant change are shown in blue (p <0.05), and the others are in red. Inset summarizes the number of SuK peptides that show significant change in KO samples. (D) Largest fold-changes (KO:WT) for individual SuK sites with the average protein expression ratio displayed. (E) Highly regulated proteins enriched with SIRT5 target sites with the average protein expression ratio displayed. Number of SIRT5 target sites in black, number of non-target sites in grey. See Dataset S3 and S4 for mass spec details of identified peptides used for protein quantification. Abbreviations include ATP synthase subunit O (ATPO), carbamoyl-phosphate synthetase 1 (CPSM), 3-ketoacyl-CoA thiolase (THIM), acyl-coenzyme A synthetase medium-chain family member 1 (ACSM1), malate dehydrogenase (MDHM), enoyl-CoA delta isomerase (ECI1), acetyl-CoA acetyltransferase (THIL), glutamate dehydrogenase (DHE3), trifunctional enzyme α subunit (ECHA), ADP/ATP translocase 2 (ADT2), 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMCS2), citrate synthase (CISY), 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), aconitase (ACON), acyl-CoA synthetase family member 2 (ACSF2), enoyl-CoA delta isomerase 2 (ECI2), GDP-specific succinyl-CoA synthetase β subunit (SUCB2), HSP10 (CH10), 3-hydroxybutyrate dehydrogenase (BDH), ADP-specific succinyl-CoA synthetase β subunit (SUCB1), 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), prostaglandin G/H synthase 2 (PHS2), and succinyl-CoA synthetase α subunit (SUCA).

Figure 3. Site distribution, conservation, and sequence logo analysis

(A) Distribution of the number of succinylation sites per protein. (B) Distribution of the number of SIRT5 target sites per protein. For peptide information and quantitation of individual sites see Dataset S1 and S2. (C) Consensus sequence logo plot for succinylation sites ± 10 amino acids from the lysine of all succinylated sites identified, (D) from the lysine of all SIRT5 target sites (> twofold and p <0.01). SIRT5 target sequence context heatmap is also shown in Figure S3A. (E) Heatmap depicting the conservation index of the SIRT5 target sites across seven vertebrate species. Percent conservation calculated for all succinylated sites, SIRT5 target sites or non-target sites is shown above the heatmap. Lysine (K), glutamine (Q), arginine (R), aspartic acid (D) or glutamic acid (E), and other amino acids are presented in different colors. Heatmap depicting the conservation index of non-target sites is available at Figure S3B. See Dataset S5 for conservation index analysis details. (F) Venn diagrams showing the overlap of succinylated and acetylated lysine residues identified in mouse liver mitochondria and the overlap of sites that are targeted by SIRT5 and SIRT3.

Figure 4. Fatty acid β-oxidation and ketone body synthesis are highly targeted by SIRT5

(A, B) Pathway analysis of succinylation (A) and SIRT5 targets (B) with the number of proteins identified per pathway. Fatty acid β-oxidation and ketone body synthesis are highlighted in blue. (C) Schematic depicting the core machinery of fatty acid β-oxidation and ketone body synthesis with SIRT5 target sites indicated on each protein. (D) Succinylation profiles of enzymes involved in fatty acid β-oxidation and ketone body synthesis. KO:WT ratio of each SuK site is shown in scatter plots. Red horizontal bar represents the median KO:WT ratio of all SuK sites on each protein. Dotted line indicates a KO:WT ratio of 2. Abbreviations include very long chain acyl-CoA dehydrogenase (ACADV), long chain acyl-CoA dehydrogenase (ACADL), medium chain acyl-CoA dehydrogenase (ACADM), short chain acyl-CoA dehydrogenase (ACADS), trifunctional enzyme α subunit (ECHA) and β subunit (ECHB), 2,4-dienoyl-CoA reductase (DECR), enoyl-CoA delta isomerase 1 (ECI1), short chain enoyl-CoA hydratase (ECHM), short chain 3-hydroxyacyl-CoA dehydrogenase (HCDH), acetoacetyl-CoA thiolase (THIL), 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMCS2), 3-hydroxy-3-methylglutarate-CoA lyase (HMGCL), and 3-hydroxybutyrate dehydrogenase (BDH).

Figure 5. Lack of SIRT5 is associated with impaired fatty acid β-oxidation

(A) Oxidation of deuterium-labeled palmitate in primary cultured hepatocytes isolated from WT (black bars) or Sirt5^−/− (blue bars) mice measured after a 24- or 48-hour incubation. Y axis represents μmoles of deuterium-labeled palmitate oxidized per 10⁶ cells (n=3). Results are shown as the mean ± standard deviation. (B) Oxidation of deuterium-labeled palmitate, when cultured in control medium, or glucose-deprived medium, or treated with 100 μM Etomoxir (ETO), measured after 24-hour incubation (n=3). Results are shown as the mean ± standard deviation. (C) Oxidation of tritium labeled palmitate in mouse embryonic fibroblasts (MEF) derived from WT (black bars) or Sirt5^−/− (blue bars) mice after a 3- or 24-hour incubation. Y axis represents nmoles of tritium-labeled palmitate oxidized per mg of proteins (3 h: n=8; 24 h: n=7). Results are shown as the mean ± standard deviation. (D) Concentrations of acylcarnitines in liver or skeletal muscle of Sirt5^−/− mice are shown as relative values compared to WT. The grey rectangles and their corresponding vertical grey lines show the chain-length-level inference (posterior mean and 95% CI). The horizontal lines and shapes show stratum-specific inference. Table below shows estimated percent increase, 95% CI, and P-value calculated for relative acylcarnitine concentrations in KO vs. WT in liver, skeletal muscle, or combined. Relative concentrations of individual acylcarnitines in KO vs. WT are also shown in Figure S4.

Figure 6. Lack of SIRT5 leads to decreased β-hydroxybutyrate production and hypersuccinylation of HMGCS2

(A) Plasma β-hydroxybutyrate levels in WT and Sirt5^−/− mice at 0, 4, 12, 16 and 24 hour following fasting (n=5, **p<0.001, *p<0.01). Results are shown as the mean ± standard error. (B) Liver β-hydroxybutyrylcarnitine levels in WT and Sirt5^−/− mice under fed or 24 hour fasted condition (n=5). Results are shown as the mean ± standard error. (C) Western blot showing succinylation of endogenous HMGCS2 immunoprecipitated from WT or Sirt5^−/− mouse liver (n=3). Integrated density values were calculated and are shown relative to WT mice. (D) Expression plasmids for WT HMGCS2 were transfected into HEK293 cells with an empty vector control, SIRT5 WT, or SIRT5 H158Y (catalytically inactive mutant). Immunoprecipitation and western blot were performed to examine succinylation levels of HMGCS2 (n=3). Integrated density values were calculated and are shown relative to empty vector control. (E) HEK293 cells over-expressing WT HMGCS2 were infected with lentivirus carrying scrambled shRNA or two different shRNAs for specifically knocking down SIRT5. Immunoprecipitation and western blot were performed to examine succinylation levels of HMGCS2 (n=5). Integrated density values were calculated and are shown relative to scrambled shRNA treatment. (F) Mass spectrum peak intensity of all identified succinyllysine-containing peptides measured in WT (black bars) and Sirt5^−/− mice (blue bars). Fold change of each site (KO:WT) is indicated above the bars.

Figure 7. HMGCS2 mutations at sites of succinylation within the substrate binding region regulate its enzymatic activity

(A) Ribbon diagram of the crystal structure of human HMGCS2 (PDB entry 2WYA) bound with HMG-CoA. Lysines fully observed in the crystal structure are shown in yellow, and lysines without fully defined conformations in the crystal structure are modeled in PyMol using low-energy all-trans rotamers as allowed by local backbone and steric interactions and displayed as green atomic spheres at 50% scale. (B) The interactions between several lysine side chains and the negatively charged CoA phosphate groups in the substrate binding pocket are shown in detail. Lysine amine nitrogens for residues 83, 306 and 310 are within 4Å of the phosphate oxygen atoms. (C) The observed mass spectrum peak intensity of succinyl-lysine-containing peptides plotted with respect to their distance to the nearest CoA phosphate. Dotted lines connect the same peptide identified in WT and Sirt5^−/− mice. (D) Steady state kinetic analysis of WT HMGCS2 and succinyllysine mimetics HMGCS2-K83E, HMGCS2-K310E, and HMGCS2-K83,310E enzymatic activity as measured by DTNB detection of CoA-SH released from increasing concentrations of acetyl-CoA at 412 nM. Graph is representative of two independent experiments, n=3 measurements/sample, mean ± SD. Inset contains values for average Km and Vmax ± SEM. The purity and equal amounts of immunoprecipitated HMGCS2 WT or mutants are shown in Figure S5.