Fig 1.
Validation of SIRT1 modulators in HEK293 cells.
(A) Chemical structures of the SIRT1 modulators EX527 (an inhibitor), SRTCX1002 (an activator) and SRTCZ1001 (a placebo). (B) HEK293 cells were treated with DMSO (blank control, I) or EX527 (II) for 48 hours or EX527 for 24 hours and then switched to SRTCX1002 (III) or SRTCZ1001 (IV) treatment for an additional 24 hours (left panel). The acetylation levels of p53 lysine-382, a known target of SIRT1 deacetylation, were examined by western blotting using a specific antibody. The p53 and SIRT1 protein levels were also determined by western blotting. β-tubulin was used as a loading control.
Fig 2.
Patterns of global protein acetylation after SIRT1 modulator treatments in 3T3-L1 mature adipocytes.
(A) Schematic illustration of the experimental procedure. Briefly, 3T3-L1 cells were differentiated into mature adipocytes for 6 days. Then, the mature adipocytes were treated with SIRT1 modulators as follows: (I) DMSO for 48 hours; (II) EX527 for 48 hours; (III) EX527 for 24 hours followed by a switch to SRTCX1002 for 24 hours; and (IV) EX527 for 24 hours followed by a switch to SRTCZ1001 for 24 hours. (B) SIRT1 protein levels were examined at the indicated times during 3T3-L1 adipogenesis by western blotting using an antibody against the C-terminal portion of SIRT1. Calnexin was used as a loading control. (C) Patterns of global protein acetylation in 3T3-L1 mature adipocytes after SIRT1 modulator treatments, as described in panel (A). The protein acetylation state was evaluated using a pan-acetyl-lysine antibody. The SIRT1 protein level was evaluated using an antibody against the C-terminal portion of SIRT1. Calnexin was used as a loading control.
Fig 3.
Proteomic analysis of SIRT1 modulator-treated 3T3-L1 mature adipocytes identified ATP6V1B2 as a potential SIRT1 target.
(A) Representative 2D-PAGE gel transfer blot stained with Coomassie blue and utilized in western blot analysis using a pan-acetyl-lysine antibody. (B) Western blot spots corresponding to ATP6V1B2 showed differential acetylation states upon SIRT1 modulator treatments, as described in Fig 2A. The spot intensities were quantified and are shown as a bar graph in the right panel. The value for the blank control was arbitrarily set as one. (C) The protein level of ATP6V1B2 was examined by western blot analyses after SIRT1 modulator treatment of 3T3-L1 mature adipocytes. Calnexin was used as a loading control.
Fig 4.
SIRT1 directly interacts with ATP6V1B2.
(A) The sub-cellular localization of SIRT1 and ATP6V1B2 was determined by western blot analyses of fractionated mitochondrial (Mito), nuclear (Nuclei), and cytosolic (Cyto) samples from 3T3-L1 mature adipocytes. Markers of the mitochondrial (HK-1), nuclear (histone H2A) and cytosolic (β-tubulin) fractions were examined by western blotting to verify the purity of our preparations. Whole-cell lysate (WCL) was included as a positive control. (B) Sub-cellular localization of SIRT1 and ATP6V1B2 was examined by immunofluorescence analysis of 3T3-L1 mature adipocytes using a SIRT1 antibody and an ATP6V1B2 antibody, respectively. Nuclei were stained with DAPI. (C) Flag-tagged full-length SIRT1 (Flag-SIRT1) was expressed in HEK293 cells and immunoprecipitated by M2-agarose. The interaction between ATP6V1B2 and Flag-SIRT1 was examined by western blot analysis of ATP6V1B2 in the Flag-IP sample. The protein levels of SIRT1, Flag-SIRT1 and ATP6V1B2 were assessed by western blotting using the corresponding antibodies. β-tubulin was used as a loading control. The asterisk indicates a non-specific band detected by the Flag antibody.
Fig 5.
SIRT1 deacetylates ATP6V1B2 in vitro and in vivo.
(A) Acetylated p53 (Ac-p53) peptide was deacetylated by SIRT1 in vitro. The deacetylation level of the peptide was evaluated by measuring the fluorescent signal from free ammonia, a byproduct of the deacetylation reaction catalyzed by sirtuin enzymes. EX527, a specific SIRT1 inhibitor, significantly reduced the ability of SIRT1 to deacetylate the Ac-p53 peptide. (B) ATP6V1B2 protein purified from wheat germ is acetylated at lysine residues. The acetylation status of ATP6V1B2 was examined using a pan-acetyl-lysine antibody in the western blot analysis. Recombinant histone H3 (rH3) was used as a negative control for lysine acetylation. The protein levels were determined by Coomassie Brilliant Blue (CBB) staining. (C) ATP6V1B2 was deacetylated by SIRT1 in vitro. The data presented in this Fig are based on three independent experiments (mean ± SED) (** P < 0.01). (D) Endogenous ATP6V1B2 was immunoprecipitated by a specific antibody in HEK293 cells with or without over-expression of His/Myc-tagged full-length SIRT1 (SIRT1-His/Myc). The state of lysine acetylation of ATP6V1B2 was examined by western blot analysis using a pan-acetyl-lysine antibody in the IP sample. The protein levels of SIRT1, SIRT1-His/Myc and ATP6V1B2 were assessed by western blotting using the corresponding antibodies. β-tubulin was used as a loading control.