title: Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine: a mechanism for the slow acetylator phenotype and substrate-dependent down-regulation.
abstract: Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives ( approximately 4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.
authors.affiliation: Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital, Perth, Western Australia 6000. n.butcher@uq.edu.au
authors.forename: Neville J
authors.initials: NJ
authors.lastname: Butcher
authors.forename: Ajanthy
authors.initials: A
authors.lastname: Arulpragasam
authors.forename: Rodney F
authors.initials: RF
authors.lastname: Minchin
mesh_terms: D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D000483:Alleles; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000378:metabolism; D005838:Genotype; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress; D010447:Peptide Hydrolases / Q000737:chemistry; D010641:Phenotype; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D007527:Isoenzymes; D007976:Leupeptins; D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D025801:Ubiquitin; D003513:Cycloheximide; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D010129:4-Aminobenzoic Acid