Lysine 88 acetylation negatively regulates ornithine carbamoyltransferase activity in response to nutrient signals

Abstract

Ornithine carbamoyltransferase (OTC) is a key enzyme in the urea cycle to detoxify ammonium produced from amino acid catabolism. OTC deficiency is an X-linked genetic disorder ranging from fatal in newborns to hyperammonemia and anorexia in adults. Through affinity purification of acetylated peptides and mass spectrometry, we identified that OTC is acetylated on lysine residues, including Lys88, which is also mutated in OTC-deficient patients. OTC acetylation was confirmed to occur under physiological conditions. Biochemical characterizations revealed that OTC Lys88 acetylation decreases the affinity for carbamoyl phosphate, one of the two OTC substrates, and the maximum velocity, whereas the K(m) for ornithine, the other OTC substrate, is not affected. Furthermore, Lys88 acetylation is regulated by both extracellular glucose and amino acid availability, indicating that OTC activity may be regulated by cellular metabolic status. Our results provide an example of the novel mechanism of regulating metabolic enzyme activity through protein acetylation.

FIGURE 1.

OTC is acetylated at Lys⁸⁸. A, shown is a tandem mass spectrum of the acetylated OTC lysine 88-containing peptide SLGMIFEK^*R. B, the acetylated Lys⁸⁸ in OTC is conserved. The sequences around OTC Lys⁸⁸ from different species were aligned. Conserved lysine residues corresponding to human OTC Lys⁸⁸ are boxed. C, transfected OTC is acetylated. HEK293T cells were transfected with pcDNA3 vector, pcDNA3-hOTC-FLAG, and pcDNA3-FLAG-p53 followed by deacetylase inhibitor treatment. Cell lysate was immunoprecipitated with FLAG beads. The precipitated OTC-FLAG was detected by anti-FLAG antibody and anti-acetyllysine antibody (α-AcK) as indicated. NAM and TSA denote treatment with nicotinamide and triochostatin A, respectively. D, endogenous OTC is acetylated. Chang liver cells were treated with deacetylase inhibitors. Endogenous OTC protein was immunoprecipitated with anti-OTC antibody. The acetylation level of endogenous OTC was probed by anti-acetyllysine antibody. E, OTC is acetylated on Lys⁸⁸. HEK293T cells were co-transfected by pcDNA3-hOTC-FLAG and pcDNA3-hOTCK88Q-FLAG. OTC proteins were precipitated by FLAG beads. OTC Lys⁸⁸ acetylation levels were detected by an antibody raised against OTC Lys⁸⁸ peptide.

FIGURE 2.

Inhibition of deacetylase reduces OTC activity. A, deacetylase inhibitors decrease transfected OTC activity. HEK293T cells were transfected with pcDNA3-hOTC-FLAG and treated with deacetylase inhibitors as indicated. OTC proteins were immunoprecipitated with FLAG beads and were eluted by 100 μl of FLAG peptide. OTC assay was carried out, and specific activity was normalized by OTC protein levels determined by Western blotting. Shown are mean ± S.D. of duplicate assays. The overall acetylation level and Lys⁸⁸ acetylation level were assayed by anti-acetyllysine antibody or anti-acetyllysine-88 antibody. B, NAM and TSA inhibit endogenous OTC activity. Chang liver cells were treated with deacetylase inhibitors. OTC protein was immunoprecipitated with anti-OTC antibody and measure the OTC activity in protein A/G beads. Antihemagglutinin (HA) antibody was used as an IP control. Bars and error bars represent mean ± S.D. of triplicate assays. Specific OTC activities were normalized by the OTC protein level.

FIGURE 3.

Acetylation of Lys⁸⁸ inhibits OTC activity. A, Lys⁸⁸ is important for full OTC activity. The wild-type OTC and K88R and K88Q mutants were expressed in HEK293T cells. Proteins were purified by IP, and OTC activity assays were determined. Relative specific OTC activities were normalized by protein level. Wild-type OTC activity was arbitrarily set as 100%. α-Hemagglutinin (HA) antibody was used as an IP control. Bars and error bars represent mean ± S.D. of triplicate assays. B, Lys⁸⁸ is required for NAM and TSA to repress OTC activity. OTC and the K88R mutant proteins were overexpressed in HEK293T cells followed by deacetylase inhibitor treatment. Proteins were purified by IP, and the total acetylation level, Lys⁸⁸ acetylation level, and OTC activities were determined, respectively. Relative specific OTC activities were normalized by protein level. α-Hemagglutinin antibody was used as an IP control. Bars and error bars represent mean ± S.D. of triplicate assays.

FIGURE 4.

Lys⁸⁸ acetylation is influenced by glucose and amino acid availability. A, glucose increases OTC acetylation and decreases activity. OTC-FLAG proteins were overexpressed in HEK293T cells cultured under different glucose concentrations as indicated. Proteins were purified by IP. OTC activity, OTC acetylation, and Lys⁸⁸ acetylation were each carried out for purified proteins. Shown are the mean ± S.D. values of triplicate assays for relative OTC activities. Enzyme activity at 0 mm amino acid was arbitrarily set as 100%. All specific activities were normalized by protein levels.B, amino acids increase OTC Lys⁸⁸ acetylation and decrease activity. OTC-FLAG proteins were overexpressed in HEK293T cells cultured under different amino acid concentrations as indicated. Proteins were purified by IP. OTC activity, OTC acetylation, and Lys⁸⁸ acetylation were each carried out for purified proteins. Mean ± S.D. values of triplicate assays for relative OTC activities were presented. Enzyme activity at 25 mm glucose was arbitrarily set as 100%. All specific activities were normalized by protein levels.