Activation of EHHADH and MDH by acetylation. (A) Acetylation of EHHADH was increased by deacetylase inhibitors. Ectopically expressed and immunoprecipitated (IP) EHHADH was examined by immunoblotting (IB) with antibody to acetyllysine (α-AcK). (B) Quantification of EHHADH acetylation by iTRAQ MS. Quantification of peptides was calculated on the basis of relative intensity of the iTRAQ tags. (C) Activation of EHHADH in cells expose to deacetylase inhibitors. Data in this panel and subsequent figures are from triplicate experiments. (D) Fatty acid induced EHHADH acetylation and activity. Acetylation and activity of EHHDH ectopically expressed in HEK293T cells were monitored. (E) MDH acetylation. MDH-Myc was expressed in HEK293T cells and acetylation was determined by immunoblotting. (F) Quantitative MS analysis of MDH. FLAG tagged MDH was overexpressed in HEK293T cells and purified by immunoprecipitation. Eluted intact MDH proteins were analyzed by FTICR MS. (G) Glucose enhances MDH acetylation. (H) Activation of MDH by acetylation. The activity of endogenous and ectopically expressed MDH from Chang and HEK293T cells, respectively, were assayed and normalized against actin. (I) Inactivation of MDH by in vitro deacetylation. Immunoprecipitated MDH was incubated with or without CobB deacetylase and activity was assayed. NAD, an essential cofactor for CobB, was omitted as a negative control. (J) Activation of MDH by glucose. Experiments were similar to (H) except cells were treated with glucose.

Destabilization of PEPCK1 by acetylation. (A) Glucose induces PEPCK1 acetylation. (B) Amino acids decrease PEPCK1 acetylation. (C) Glucose induces depletion in of PEPCK1 protein. Endogenous PEPCK levels were detected with a PEPCK1 antibody. (D) TSA and NAM reduce PEPCK1 protein abundance. (E) Glucose destabilizes PEPCK1. Cycloheximide was added at time zero to block translation in HEK293 cells. PEPCK1 protein abundance was determined by Western blotting. (F) Inhibition of deacetylases destabilizes the wild type but not the acetylation-defective mutant PEPCK1.

Acetylation of liver metabolic enzymes. (A) Comparison of three acetylation proteomic studies: this study and (2, 3). (B) Preferential acetylation of enzymes in intermediary metabolism. Fisher’s exact test of comparing acetylated proteins to total liver proteins shows that acetylation is much more prevalent in intermediary metabolic enzymes. (C to G) Acetylated metabolic enzymes identified by proteomic survey are marked in red. See supporting online material for key to abbreviations.

Inactivation of ASL by acetylation. (A and B) ASL acetylation. FLAG-tagged ASL or ASL^K288R was overex-pressed in HEK293T cells, immunoprecipitated, and probed with antibody to acetyllysine (A) or to acetyl-Lys²⁸⁸ (B). (C) Inhibition of ASL acetylation by extra amino acids. ASL was immunoprecipitated from transfected HEK293T cells, which were treated with various amino acid concentrations. (D) Inhibition of ASL in response to NAM and TSA. Wild-type and mutant ASL-K288R (132% of wild-type activity) proteins were expressed in HEK293T cells that were treated with NAM and TSA as indicated. Activity of immuno-precipitated ASL was normalized to total protein. (E) Requirement of Lys²⁸⁸ acetylation for ASL activation by amino acids. Wild-type and mutant ASL proteins were overexpressed in HEK293T cells incubated in medium containing various amino acid concentrations. (F and G) Effects of glucose on acetylation and activity of ASL. ASL was overexpressed in HEK293T cells that were treated with various concentrations of glucose. Acetylation and activity of immunoprecipitated ASL were determined. (H) Activation of ASL by in vitro deacetylation. In vitro deacetylation was similar to Fig. 2I.