The Acetylation Of Metabolic Enzymes Proteomics And Ornithine Carbamoyl Transfer The Regulation Of Enzyme Acetylation

Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation.The majority of acetylation studies have been focused on histones and nuclear transcription regulators,and have revealed a fundamental importance of acetylation in transcription regulation.In fact,most transcription activators or repressors function to recruit histone acetyltransferases and deacetylases,respectively,to modulate target gene expression.Here the first charpter is a review of the posttranslational acetylation of non-histone proteins,which take part in cellular regulatory processes.The histone acetyltransferases and deacetylases recruit subunits to compose the complexes which regulate cellular proliferation and differentiation.With growing number of identified acetylated non-histone proteins demonstrate that the reversible lysine acetylation affects mRNA stability,and the localization, interaction,degradation of proteins.It is worthy noting the acetylated proteins are involved in tumourigenesis,cancer cell proliferation,cellular senescence, apoptosis and immune reactions.So inhibitors of histone deacetylases could be considered as the potential drugs for human diseases therapy.The second charpter,The affinity-purified acetylated peptides from human liver tissues were analyzed by tandem liquid chromatography-tandem mass spectrometry(LC/LC-MS/MS).We show that lysine acetylation is a prevailing form of modification in intermediate metabolic enzymes.We identified more than 1200 acetylated peptides with high confidence MS scores.These peptides match to 978 distinct human proteins.Kim et al previously reported a mouse proteomic analysis that identified 195 acetylated proteins and 114(58%) of which were also present in our proteomic analysis.Virtually every enzyme in glycolysis,gluconeogenesis,tricarboxylic acid(TCA)cycle,urea cycle,fatty acid metabolism,and glycogen metabolism are found to be acetylated in human liver tissue.Furthermore,metabolic fuels,such as glucose,amino acids,and fatty acids,regulate acetylation status of metabolic enzymes.The third charpter,we selected Ornithine carbamoyltransferase(OTC)to elucidate the acetylation how to regulate OTC's function.OTC is a key enzyme in the urea cycle to detoxify ammonium produced from amino acids 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 K88,which is also mutated in OTC deficiency patients.OTC acetylation was confirmed to occur under physiological conditions.Biochemical characterizations revealed that OTC K88 acetylation decreases the affinity for carbamoyl phosphate,one of the two OTC substrates,and the maximum velocity while the Km for ornithine,the other OTC substrate,is not affected.Furthermore,K88 acetylation is regulated by both extracellular glucose and amino acid availability,indicating that OTC activity may be regulated by cellular metabolic status.Next we found SIRT3 is the deacetylase to OTC and deacetylate to ac-K88.Our results provide an example of novel mechanism of regulating metabolic enzyme activity through protein acetylation.Our findings reveal a previously unrecognized general role of acetylation in cellular metabolic regulation.Importantly,in all of the representative enzymes we have studied,acetylation levels of each enzyme change in response to the changes of extracellular nutrient availability,providing evidence for a physiological role of acetylation in metabolic regulation.We propose that lysine acetylation is an evolutionally conserved mechanism involved in metabolic regulation in response to the availability of different nutrient sources and cellular metabolic status.Acetylation may play a key role in the coordination of different metabolic pathways in response to different extracellular environments.Further studies to determine the regulation of acetylation,the responsible acetyltransferases and deacetylases,and the molecular mechanisms of acetylation on target protein functions should provide new insights into physiological regulation of metabolism and pathological alterations in diseases.