Fig. 6: NAT10 acetylates ACLY at lysine 468 to stabilize ACLY.

A In vitro acetylation was performed with purified GST-ACLY and Flag-NAT10 proteins with or without acetyl-CoA. The reaction products were detected by WB. The purified GST-ACLY used in the experiment was shown by Coomassie blue staining. B Huh7-NAT10 shRNA-1, Huh7-NAT10 shRNA-2, and Huh7-control shRNA cells were subjected to immunoprecipitation using anti-acetyl-lysine. The acetylation levels of ACLY were evaluated by WB using anti-ACLY. C Cells were transfected with indicated plasmids. Proteins were immunoprecipitated using anti-acetyl-lysine and the acetylation levels of ACLY were evaluated by WB using anti-ACLY. D In vitro acetylation experiment was performed with indicated proteins and acetyl-CoA. The acetylation levels of GST-ACLY truncated proteins were evaluated by WB using anti-acetyl-lysine. The amounts of GST fusion proteins used in the experiments were shown by Coomassie blue staining. E In vitro acetylation experiment was performed as (A) and the reaction products were resolved by SDS-PAGE. The band of acetylated ACLY was cut, fully trypsinized, and analyzed by LC–MS/MS (Thermo). Mass spectrometry data were processed using the Proteome Discoverer software (Version 1.4). F In vitro acetylation experiment was performed with indicated proteins and acetyl-CoA. The acetylation levels of GST-ACLY mutant proteins were evaluated by WB using anti-acetyl-lysine. The amounts of GST fusion proteins used in the experiments were shown by Coomassie blue staining. G Cells were transfected with indicated plasmids. Proteins were immunoprecipitated using anti-acetyl-lysine and the acetylation levels of ACLY were evaluated by WB using anti-GFP. H Cells were transfected with indicated plasmids and treated with CHX at indicated time points. Proteins from cell lysates were immunoblotted with the antibodies as indicated (upper panel). Relative GFP-ACLY protein levels at different time points were shown (lower panel). Data were analyzed by one-way ANOVA and presented as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I Cells were transfected with indicated plasmids and treated with 10 μM MG132. Then lysates were subjected to immunoprecipitation using anti-GFP. The ubiquitination levels of ACLY were evaluated by WB using anti-HA. J Venn diagram showed the common proteins between the ACLY’s E3 ubiquitin ligase including UBR4, Cullin3 (CUL3), Neuronally expressed developmentally down-regulated 4 (NEDD4), SQSTM1, Hrd1, and the Flag-NAT10-binding proteins in HeLa cells [28]. The blue area illustrates the number of Flag-NAT10-binding proteins, the red area illustrates the number of ACLY’s E3 ubiquitin ligase, and the gray area illustrates the number of common proteins between two datasets. K Huh7 cells were treated with 10 μM oxaliplatin. The cytoplasmic lysate and nuclear extraction were fractioned, which were subjected to immunoprecipitation using anti-ACLY antibody. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. TOP I and α-tubulin were used as nuclear and cytoplasmic marker, respectively. L Huh7 cells were transfected with indicated plasmids. Proteins were immunoprecipitated using anti-GFP. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. M Schematic model of ACLY protein level regulated by SQSTM1 under unstressed or DNA damage stress conditions. ACLY binds to SQSTM1 and is degraded by SQSTM1-mediated ubiquitination under unstressed condition. However, ACLY is acetylated by NAT10 after DNA damage which prevents the binding between ACLY and SQSTM1, thus leading to increased ACLY protein level. N Huh7-control shRNA and Huh7-NAT10 shRNA-2 cells were transfected with control siRNA or SQSTM1 siRNA and then harvested at indicated time points after treatment with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative ACLY protein levels at indicated time points were shown (right panel). Data were analyzed by one-way ANOVA and presented as mean ± SEM (n = 3), *P < 0.05, **P < 0.01.