1. Volume 19, Issue 11, Pages 2331-2344 (June 2017)
SIRT5 Desuccinylates and Activates Pyruvate Kinase M2 to Block Macrophage IL-1β Production and to Prevent DSS-Induced Colitis in Mice 
Fang Wang, Ke Wang, Wei Xu, Shimin Zhao, Dan Ye, Yi Wang, Ying Xu, Lisha Zhou, Yiwei Chu, Cuiping Zhang, Xue Qin, Pengyuan Yang, Hongxiu Yu 
Cell Reports 
Volume 19, Issue 11, Pages (June 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 2331-2344DOI: (10.1016/j.celrep.2017.05.065)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1
SIRT5 Interacts with and Desuccinylates PKM2 Activity, and SIRT5-Dependent Succinylation Promotes PKM2 Dimerization and Increases Its Protein Kinase Activity
(A) Tagged SIRT5 interacted with and tagged PKM2. HEK293T cells were transfected with indicated plasmids, and PKM2-SIRT5 association was examined by co-immunoprecipitation and western blotting.
(B) Endogenous SIRT5 bound with endogenous PKM2. Endogenous co-immunoprecipitation of SIRT5 and PKM2 was performed as indicated in the lysates of HEK293T cells, and the association was examined by western blotting.
(C and D) SIRT5 desuccinylated Flag-PKM2, but it did not affect its malonylation and glutarylation. HEK293T cells with overexpression (C) or knockdown of SIRT5 (D) and its control cells were transfected with Flag-PKM2, followed by immunoprecipitation. Succinylation, glutarylation, and malonylation of PKM2 were determined by western blotting and normalized to its protein level.
(E and F) PKM2 was highly succinylated by succinyl-CoA in vitro. Recombinant(r)PKM2 (rPKM2) purified from E. coli and Flag-PKM2 overexpressed and immunoprecipitated from HEK293T cells were treated with succinyl-CoA (1 mM) for 15 min. Pyruvate kinase activity was determined followed by western blotting using an anti-pan-succinylysine antibody. (E) Total protein level of PKM2 was detected by Coomassie blue staining. (F) Total protein level of PKM2 was detected by western blotting.
(G) SIRT5 knockdown decreased pyruvate kinase activity of Flag-PKM2. HEK293T cells with SIRT5 knockdown and its control cells were transfected with Flag-PKM2, followed by immunoprecipitation. Pyruvate kinase activity of Flag-PKM2 was detected and normalized to its protein level.
(H) SIRT5 desuccinylated and activated Flag-PKM2 in vitro. HEK293T cells were transfected with indicated plasmids, followed by immunoprecipitation. Immunoprecipitated Flag-PKM2 was incubated with His-SIRT5 in vitro. Pyruvate kinase activity and succinylation level of PKM2 were determined.
(I) SIRT5 overexpression increased pyruvate kinase activity of Flag-PKM2. HEK293T cells with SIRT5 overexpression and its control cells were transfected with Flag-PKM2, followed by immunoprecipitation. Pyruvate kinase activity of Flag-PKM2 was detected and normalized to its protein level.
(J) SIRT5 knockdown promoted tetramer-to-dimer transition of Flag-PKM2. HEK293T cells with SIRT5 knockdown and its control cells were transfected with Flag-PKM2, and their interaction with endogenous PKM2 was analyzed by immunoprecipitation and western blotting.
(K) SIRT5 knockdown prevented tetramerization of Flag-PKM2. HEK293T cells with SIRT5 knockdown and its control cells were transfected with Flag-PKM2. Cell lysates were prepared, separated by gel filtration, followed by western blotting for PKM2 protein.
(L) Succinyl-coA promoted protein kinase activity of rPKM2 in vitro. rPKM2 was pre-treated with succinyl-CoA (1 mM) or untreated for 15 min and then incubated with purified histone H3. Protein kinase activity of rPKM2 was determined by western blotting using antibody against Thr11- or Ser10-phosphorylated histone H3.
(M) SIRT5 knockdown promoted protein kinase activity of PKM2. The total lysates of HEK293T cells with SIRT5 knockdown and its control cells were analyzed by western blotting using antibody against Thr11-and Ser10-phosphorylated histone H3.
Data represent means ± SE. ∗p < 0.05, ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Lys311 Succinylation Promotes PKM2 Tetramer-to-Dimer Transition and Protein Kinase Activity
(A and B) Seven confidential succinylated sites were identified in Flag-PKM2. HEK293T cells were transfected with indicated plasmids, followed by immunoprecipitation and analysis by LC-MS/MS. (A) Succinylated sites detected. (B) MS spectrum of the succinylated site Lys311.
(C) Lys311 and Lys498 were highly conserved in different species, ranging from Xenopus to Human.
(D) K311 mutation decreased pyruvate kinase activity of PKM2. HEK293T cells were transfected with indicated plasmids, followed by immunoprecipitation. Pyruvate kinase activity and succinylation level of PKM2 were normalized to its protein level.
(E) Succinyl-CoA decreased pyruvate kinase activity of WT PKM2 while not affecting that of K311E or K311E/K498E mutants. HEK293T cells were transfected with indicated plasmids, followed by immunoprecipitation and treatment with or without succinyl-CoA (1 mM) for 15 min. Pyruvate kinase activity and succinylation level of PKM2 were normalized to its protein level.
(F) The K311E mutant totally abolished its interaction with endogenous PKM2. HEK293T cells with SIRT5 knockdown and its control cells were transfected with indicated plasmids, and their interaction with endogenous PKM2 was analyzed by immunoprecipitation and western blotting.
(G) The K311E mutant showed a higher level of nuclear accumulation. HEK293T cells with SIRT5 knockdown and its control cells were transfected with indicated plasmids. The nuclear fractions and cytosol fractions were prepared and analyzed by western blotting.
(H) The K311E mutant displayed significantly stronger protein kinase activity to T11 of histone H3 in an in vitro assay. HEK293T cells were transfected with indicated plasmids, followed by immunoprecipitation and incubation with recombinant histone H3. Protein kinase activity of PKM2 was determined by western blotting using antibody against Thr11-phosphorylated histone H3.
(I) The K311E mutant showed increased protein kinase activity in SIRT5-knockdown HEK293T cells. HEK293T cells with SIRT5 knockdown and its control cells were transfected with indicated plasmids. The total cell lysates were prepared and analyzed by western blotting.
Data represent means ± SE. ∗p < 0.05, ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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5. Figure 3
SIRT5 Regulates PKM2 Activity and Modulates IL-1β Production in LPS-Activated Macrophages
(A) Secreted IL-1β was markedly increased in LPS-induced Sirt5 KO macrophages. The culture medium of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and then determined by ELISA.
(B) Protein expression of IL-1β was markedly increased in LPS-induced Sirt5 KO macrophages. The total cell lysates of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and then determined by western blotting; β-actin was used as a loading control.
(C) IL-1β transcription level was markedly increased in LPS-induced Sirt5 KO macrophages. The total mRNA of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and determined by qRT-PCR; β-actin was used as a loading control.
(D) IL-6 and TNF-α transcription level were markedly increased in LPS-induced Sirt5 KO macrophages. The total mRNA of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and determined by qRT-PCR; β-actin was used as a loading control. (left) IL-6 mRNA expression; (right) TNF-α mRNA expression.
(E) IL-10 transcription level was not affected in LPS-induced Sirt5 KO macrophages. The total mRNA of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and determined by qRT-PCR; β-actin was used as a loading control.
(F) Activation of PKM2 with TEPP-46 inhibited mRNA expression of IL-1β in LPS-induced Sirt5 KO macrophages. WT and Sirt5 KO macrophages were pretreated dimethylsulfoxide or TEPP-46 for 30 min, followed by stimulation with LPS (100 ng/mL) for indicated times. The total mRNA was prepared, determined by qRT-PCR, and normalized against β-actin.
(G) Activation of PKM2 with TEPP-46 did not affect mRNA expression of IL-6, TNF-α, and IL-10 in LPS-induced Sirt5 KO macrophages. WT and Sirt5 KO macrophages were pretreated dimethylsulfoxide or TEPP-46 for 30 min, followed by stimulation with LPS (100 ng/mL) for indicated times. The total mRNA was prepared, determined by qRT-PCR, and normalized against β-actin. (left) IL-6 mRNA expression; (middle) TNF-α mRNA expression; (right) IL-10 mRNA expression.
(H) The pyruvate kinase activity of PKM2 was inhibited in LPS-induced Sirt5 KO macrophages. The total cell lysates of WT and Sirt5 KO macrophages with or without LPS treatment for indicated times were prepared, and the pyruvate kinase activity of PKM2 was determined.
(I) PKM2 was enriched in the nucleus of LPS-activated Sirt5 KO macrophages. WT and Sirt5 KO macrophages were treated by LPS (100 ng/mL) for indicated times, and subcellular localization of PKM2 was examined by immunofluorescence microscopy.
(J) The increase of low molecular weight PKM2 was found in LPS-activated Sirt5 KO macrophages compared to WT cells. WT and Sirt5 KO macrophages were treated by LPS (100 ng/mL) for indicated times, and glutaraldehyde cross-linking study was then performed, followed by western blotting to detect endogenous PKM2.
(K) Binding of PKM2 and HIF1α to the IL-1β promoter was enhanced in LPS-induced Sirt5 KO macrophages. ChIP-PCR using PKM2 and HIF1α antibodies and primers specific for −300 position of IL-1β were performed in WT and Sirt5 KO macrophages with LPS (100 ng/mL) treatment for indicated times.
(L) PKM2 mRNA level was not changed between LPS-induced WT and Sirt5 KO macrophages. The total mRNA of WT and Sirt5 KO macrophages with or without LPS (100 ng/mL) treatment for indicated times were prepared and determined by qRT-PCR; β-actin was used as a loading control.
(M) PKM2 protein level was not changed between LPS-induced WT and Sirt5 KO macrophages. The total cell lysates of WT and Sirt5 KO macrophages with LPS (100 ng/mL) treatment for indicated times were prepared, and PKM2 protein expression was determined by western blotting; β-actin was used as a loading control.
Data represent means ± SE. ∗p < 0.05, ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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6. Figure 4 Sirt5 Inhibits DSS-Induced Colitis in Mice
(A) Schematic representation of experimental procedure: WT (n = 9) and Sirt5 KO (n = 11) mice were given 2.5% (w/v) DSS in drinking water for 7 days, followed by regular drinking water for 2 days.
(B) Body-weight changes of WT and Sirt5 KO mice with or without DSS during the experiment.
(C) Colon-length changes of WT and Sirt5 KO mice with or without DSS.
(D) Statistics of colon length of WT and Sirt5 KO mice with or without DSS.
(E) Histopathological changes in colon tissue of WT and Sirt5 KO mice with or without DSS.
(F) Semiquantitative scoring of histopathology in colon tissue of WT and Sirt5 KO mice with or without DSS.
(G) IL-1β mRNA expression in colon tissue of WT and Sirt5 KO mice with or without DSS determined by qRT-PCR.
(H) IL-6 and TNF-α mRNA expression in colon tissue of WT and Sirt5 KO mice with or without DSS determined by qRT-PCR.
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7. Figure 5
Both Activation of PKM2 and Neutralization of IL-1β In Vivo Confer Protection against DSS-Induced Colitis in Sirt5-Deficiency Mice
(A–H) Activation of PKM2 in vivo conferred protection against DSS-Induced colitis in Sirt5-deficiency mice.
(A) Schematic representation of experimental procedure: WT (n = 7) and Sirt5 KO (n = 6/group) mice were fed 2.5% DSS solution in drinking water for 7 days, followed by regular drinking water for 2 days. WT mice were given vehicle, and Sirt5 KO mice were randomly divided into two cohorts, one given vehicle and the other TEPP-46 at 10 mg/kg daily throughout the duration of the experiment.
(B) Body-weight changes of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46 (10 mg/kg) during the experiment.
(C) Colon-length changes of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46.
(D) Statistics of colon length of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46.
(E) Histopathological changes in colon tissue of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46.
(F) Semiquantitative scoring of histopathology in colon tissue of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46.
(G) mRNA expression of IL-1β and Sirt5 in colon tissue of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46 determined by qRT-PCR.
(H) mRNA expression of IL-6, TNF-α, and Sirt5 in colon tissue of DSS-fed WT and Sirt5 KO mice given vehicle or TEPP-46 determined by qRT-PCR.
(I–N) Neutralization of IL-1β in vivo conferred protection against DSS-induced colitis in Sirt5-deficiency mice.
(I) Schematic representation of experimental procedure: WT (n = 12) and Sirt5 KO (n = 6/group) mice were fed 2.5% DSS solution in drinking water for 5 days, followed by regular drinking water for 2 days. Either control IgG or anti-IL-1β neutralizing antibody was administered intraperitoneally (100 μg each) on day 0, day 3, day 4, day 5, and day 6 of DSS treatment in WT and Sirt5 KO mice.
(J) Body-weight changes of DSS-fed WT and Sirt5 KO mice administered with IgG or anti-IL-1β neutralizing antibody during the experiment.
(K) Colon-length changes of DSS-fed WT and Sirt5 KO mice administered with IgG or anti-IL-1β neutralizing antibody.
(L) Statistics of colon length of DSS-fed WT and Sirt5 KO mice administered with IgG or anti-IL-1β neutralizing antibody.
(M) Histopathological changes in colon tissue of DSS-fed WT and Sirt5 KO mice administered with IgG or anti-IL-1β neutralizing antibody.
(N) Semiquantitative scoring of histopathology in colon tissue of DSS-fed WT and Sirt5 KO mice administered with IgG or anti-IL-1β neutralizing antibody.
Data represent means ± SE. ∗p < 0.05, ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
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