Figure 1.
APE1/Ref-1 regulates SIRT1 activity in vitro and in vivo.
(A) APE1/Ref-1 increases SIRT1 activity in endothelial cells. APE1/Ref1 was adenovirally over-expressed (AdAPE1/Ref-1) or knocked down with siRNA (siAPE1/Ref-1) in HUVEC. Inert adenovirus AdLacZ or scrambled siRNA (scr) were used as controls. Deacetylase activity of immunoprecipitated endogenous SIRT1 was measured. * P<0.05, # p>0.05, (n = 3). (B) Redox-deficient APE1/Ref-1 does not increase SIRT1 activity. HEK293 cells were transfected with APE1/Ref-1 (WT) or the redox-deficient APE1/Ref-1 (C65A/C93A) mutant. Deacetylase activity of immunoprecipitated endogenous SIRT1 was measured. * P<0.05, (n = 5). (C) Knockdown of APE1/Ref-1 decreases SIRT1 activity in HEK293 cells. Cells were transfected with APE1/Ref-1 siRNA or scrambled (scr) siRNA. Deacetylase activity of immunoprecipitated endogenous SIRT1 was measured. * P<0.05, (n = 5). (D) APE1/Ref-1 stimulates deacetylase activity of SIRT1 in vitro. Recombinant SIRT1 was incubated with recombinant wild-type (WT) or redox-deficient APE1/Ref-1 (C65A/C93A). Deacetylase activity was measured in the incubation mix. * P<0.05, (n = 5). In all experiments, deacetylase activity of recombinant SIRT1, or SIRT1 immunoprecipitated from cell lysates, was measured using the Fluor de Lys® Substrate and is expressed in arbitrary fluorescence units (AFU).
Figure 2.
APE1/Ref-1 increases free thiol content of SIRT1.
(A) APE1/Ref-1 reduces thiol residues of SIRT1 in endothelial cells. APE1/Ref1 was adenovirally over-expressed (AdAPE1/Ref-1) or knocked down with siRNA (siAPE1/Ref-1) in HUVEC. Inert adenovirus AdLacZ or scrambled siRNA (scr) were used as controls. * P<0.05, (n = 3). (B) Redox-deficient APE1/Ref-1 does not reduce thiol residues of SIRT1 or stimulate SIRT1 activity. HEK293 cells were transfected with APE1/Ref-1 (WT) or the redox-deficient APE1/Ref-1 (C65A/C93A) mutant. * P<0.05, # p>0.05, (n = 3). (C) Knockdown of APE1/Ref-1 decreases free thiol content of SIRT1 in HEK293 cells. Cells over-expressing SIRT1 were transfected with APE1/Ref-1 siRNA or scrambled (scr) siRNA. * P<0.05, (n = 3). (D) Cysteines 371 and 374 in the catalytic core domain of SIRT1 are targeted for reduction by APE1/Ref-1. Recombinant wild-type SIRT1 (WT) and SIRT1 mutated at cysteines 371 and 374 (C371S/C374S) were incubated with wild-type APE1/Ref-1 (WT) or redox-deficient APE1/Ref-1 (C65A/C93A) in vitro. Reduced cysteines in SIRT1 and total SIRT1 protein were densitometrically quantified. * P<0.05, (n = 3) (E) APE1/Ref-1 does not reduce thiol residues in SIRT1 mutated at cysteines 371 and 374 in HEK 293 cells. Wild-type (WT) SIRT1 and SIRT1 (C371S/374S), with and without APE1/Ref-1, were expressed in cells. In all experiments, free thiol content of SIRT1 was determined using a modified biotin-switch assay, and is expressed after normalization with total SIRT1.
Figure 3.
APE1/ref-1 protects endothelial SIRT1 from oxidative stress-induced inactivation and promotes de-acetylation of endothelial nitric oxide synthase.
(A) H2O2-induced decrease in SIRT1 activity is rescued by APE1/Ref-1. APE1/Ref1 was adenovirally over-expressed (AdAPE1/Ref-1) or knocked down with siRNA (siAPE1/Ref-1) in HUVEC. Inert adenovirus AdLacZ or scrambled siRNA (scr) were used as controls. Cells were treated with H2O2 for 30 min. * P<0.05, # p>0.05, (n = 3). (B) H2O2-induced decrease in free thiol content of SIRT1 is rescued by APE1/Ref-1. APE1/Ref1 was adenovirally over-expressed (AdAPE1/Ref-1) or knocked down with siRNA (siAPE1/Ref-1) in HUVEC. Inert adenovirus AdLacZ or scrambled siRNA (scr) were used as controls. Cells were treated with H2O2 for 3 hrs. * P<0.05, # p>0.05, (n = 3). (C) H2O2 dose-dependently inactivates SIRT1 in vitro. Recombinant active SIRT1 was treated with H2O2 for 1 hour. * P<0.05, (n = 3) In A-C, deacetylase activity of recombinant SIRT1, or SIRT1 immunoprecipitated from cell lysates, was measured using the Fluor de Lys® Substrate and is expressed in arbitrary fluorescence units (AFU). Free thiol content of SIRT1 was determined using a modified biotin-switch assay, and is expressed after normalization with total SIRT1. (D) APE1/Ref-1 rescues SIRT1 from H2O2–induced inactivation in vitro. Recombinant active SIRT1 was treated with H2O2 for 1 hour. * P<0.05, # p>0.05, (n = 3). (E) APE1/Ref-1 stimulates lysine de-acetylation of endothelial nitric oxide synthase (eNOS). APE1/Ref1 was adenovirally over-expressed (AdAPE1/Ref-1) or knocked down with siRNA (siAPE1/Ref-1) in HUVEC. Inert adenovirus AdLacZ or scrambled siRNA (scr) were used as controls. Acetylated lysine was assessed and quantified in immunoprecipitated eNOS. Values are expressed as fold change in acetylated eNOS after normalization with total eNOS. * P<0.05, # p>0.05, (n = 3). (F) Non-reducible SIRT1 inhibits APE1/Ref-1-stimulated deacetylation of eNOS. APE1/Ref-1, with or without non-reducible SIRT1 (C371S/C374S) was expressed in HUVEC. Acetylated eNOS was assessed in acetyl-lysine immunoprecipitates. * P<0.05 compared to control, and ** P<0.01 compared to APE1/Ref-1+ SIRT1 (C371S/C374S), (n = 3).
Figure 4.
SIRT1 activity and free thiol content is diminished in APE1/Ref-1 heterozygous mice.
(A) APE1/Ref-1+/− mice have decreased tissue SIRT1 activity. SIRT1 activity was measured in liver, kidney and aorta of APE1/Ref-1+/− mice and APE1/Ref-1+/+ mice. * P<0.05, (n = 3). (B and C) SIRT1 expression is unchanged in APE1/Ref-1+/− mice. SIRT1 mRNA (B) and protein (C), and APE1/Ref-1 protein (C) was assessed and quantified in liver, kidney, heart and aorta of APE1/Ref-1+/+ mice and APE1/Ref-1+/− mice. mRNA was normalized to GAPDH, and protein to β-actin, and both expressed as fold relative to APE1/Ref-1+/+ mice. * P<0.05, # p>0.05, (n = 3). (D) Free thiol content of tissue SIRT1 is diminished in APE1/Ref-1+/− mice. Free thiol content was assessed and densitometrically quantified in kidneys of APE1/Ref-1+/+ and APE1/Ref-1+/− mice. * P<0.05, (n = 3). (E) APE1/Ref-1+/− mice have increased lysine acetylation of endothelial p53. Immunohistochemistry for lysine acetylated p53 was performed in sections of aortas from APE1/Ref-1+/+ and APE1/Ref-1+/− mice. Magnification is 40×. Percentage of endothelial cells positive for detectable lysine acetylated p53 was quantified in 20 cells from three separate fields. * P<0.05, (n = 3). Arrows: positive cells identified in the endothelium and media. Deacetylase activity of SIRT1 immunoprecipitated from nuclear extracts of homogenized tissues was measured using the Fluor de Lys® Substrate and is expressed in arbitrary fluorescence units (AFU). Free thiol content of SIRT1 in nuclear extracts was determined using a modified biotin-switch assay, and is expressed after normalization with total SIRT1.
Figure 5.
SIRT1 rescues impaired endothelium-dependent vasorelaxation in APE1/Ref-1 heterozygous mice.
(A) Adenoviral gene transfer of SIRT1 increases vascular SIRT1 activity. Wild-type SIRT1 was overexpressed ex vivo in aortic sections of APE1/Ref-1+/− mice using AdSIRT1. AdLacZ was used as control. SIRT1 protein and activity in whole aortas was determined. * P<0.05, (n = 3) (B) Overexpression of SIRT1 restores endothelium-dependent vasorelaxation in APE1/Ref-1+/− mice. Wild-type SIRT1 was overexpressed ex vivo in aortic sections of APE1/Ref-1+/− mice using AdSIRT1. AdLacZ was used as control. Acetylcholine-induced endothelium-dependent vasorelaxation was measured in aortic sections of APE1/Ref-1+/+ mice (♦), APE1/Ref-1+/− mice infected with AdSIRT1 (▪), and APE1/Ref-1+/− mice infected with AdLacZ (▴).* P<0.01 compared with APE1/Ref-1+/+ and APE1/Ref-1+/−+AdSIRT1 (n = 6). (C) Bioavailable NO in aortic sections of APE1/Ref-1+/+ mice (♦), APE1/Ref-1+/− mice infected with AdSIRT1 (▪), and APE1/Ref-1+/− mice infected with AdLacZ (▴).*P<0.01 compared with APE1/Ref-1+/+ mice and APE1/Ref-1+/− mice+AdSIRT1 (n = 5). (D) Sodium nitroprusside (SNP)-induced endothelium-independent vasorelaxation in aortic sections of APE1/Ref-1+/+ mice (♦), APE1/Ref-1+/− mice infected with AdSIRT1 (▪), and APE1/Ref-1+/− mice infected with AdLacZ (▴) (n = 4).
Figure 6.
Schematic showing oxidation of vicinal thiols in the zinc tetra-thiolate center of the catalytic domain of SIRT1, and their reduction by APE1/Ref-1.