Fig. 4: p53 is succinylated at Lysine 120, while its desuccinylation was mediated by SIRT5.

A qPCR analysis of MDM2 mRNA in SIRT5-deficient HCT116 cells (SIRT5^−/−) transfected with the control plasmid (Flag empty) or the plasmid expressing Flag–SIRT5 (Flag-SIRT5) or its enzyme-deficient mutant H158Y (Flag-SIRT5-H158Y) for 24 h, followed by treatment with or without Dox (1 μM, 24 h). DMSO was used as control. Data show mean ± SEM; Student’s two-tailed t-test; Data from three independent experiments. B qPCR analysis of PIG3 mRNA in SIRT5-deficient HCT116 cells (SIRT5^−/−) transfected with the control plasmid (Flag empty) or the plasmid expressing Flag–SIRT5 (Flag-SIRT5) or its enzyme-deficient mutant H158Y (Flag-SIRT5-H158Y) for 24 h, followed by treatment with or without Dox (1 μM, 24 h). DMSO was used as control. Data show mean ± SEM; Student’s two-tailed t-test; Data from three independent experiments. C p53 was succinylated in vitro. HA-tagged p53 protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; HA-p53 was detected with anti-HA antibody. D p53 is desuccinylated by SIRT5 in vitro. Succinylated p53 was incubated with purified SIRT5. Protein succinylation was detected with anti-pan-succinyl-lysine antibody. E Disruption of SIRT5 in HCT116 cells enhanced succinylation of p53. The cell lysates from SIRT5-deficient (SIRT5^−/−) or SIRT5-intact (SIRT^+/+) HCT116 cells treated with Dox (1 μM, 24 h) were immunoprecipitated with anti-p53 antibody, followed by immunoblotting with anti-pan-succinyl-K antibody. TCL total cell lysate, IP immunoprecipitating. F Reconstitution of wild-type SIRT5 (Myc-SIRT5) in SIRT5-deficient HCT116 cells (SIRT5^−/−) caused a reduction of p53 succinylation; but overexpression of the enzymatic-deficient SIRT5 (Myc-dSIRT5-H158Y) in SIRT5^−/− HCT116 cells did not cause a reduction of p53 succinylation. The SIRT5^−/− HCT116 cells were transfected with Myc-SIRT5 or Myc-SIRT5-H158Y and treated with Dox (1 μM, 24 h), followed by immunoprecipitating with anti-p53 antibody, and immunoblotting with anti-pan-succinyl-K antibody. TCL total cell lysate, IP immunoprecipitating. G The succinylated residue in p53 identified by mass spectrometry analysis. HEK293T cells were transfected with HA-p53 plasmid. Cell lysate was immunoprecipitated with anti-HA Ab–conjugated agarose beads overnight. Immunoprecipitated p53 proteins were subjected to 8% SDS-PAGE gel, and p53 bands were excised from the gel and analyzed by mass spectrometry. H Sequence alignment of partial p53 proteins (116–132 amino acids) from human, macaque, cow, pig, dog, mouse, and zebrafish. The red box indicates a conserved lysine (K120). I Dot-blot assay for validating the specificity of anti-Su-p53-K120 antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-Su-p53-K120 antibody. J Disruption of SIRT5 in HCT116 cells caused an enhancement of p53 succinylation at Lys 120 compared with that in the SIRT5-intact HCT116 cells (SIRT5^+/+) treated with or without Dox. The cell lysates from SIRT5-deficient (SIRT5^−/−) or SIRT5-intact (SIRT5^+/+) HCT116 cells treated with or without Dox (1 μM, 24 h) were immunoprecipitated with anti-p53 antibody, followed by immunoblotting with anti-Su-p53-K120 antibody. DMSO was used as a control. TCL total cell lysate, IP immunoprecipitating. K Disruption of SIRT5 in HCT116 cells caused an enhancement of p53 succinylation at K120 compared with that in the SIRT5-intact HCT116 cells (SIRT5^+/+) treated with or without 5-Fu. The cell lysates from SIRT5-deficient (SIRT5^−/−) or SIRT5-intact (SIRT5^+/+) HCT116 cells treated with or without 5-Fu (5 μg/ml, 24 h) were immunoprecipitated with anti-p53 antibody, followed by immunoblotting with anti-Su-p53-K120 antibody. DMSO was used as a control. TCL total cell lysate, IP immunoprecipitating.