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
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
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DOI：
10.1074/jbc.m312858200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Minchin, RF
中科院分区：
文献类型：
--
作者：
Butcher, NJ;Arulpragasam, A;Minchin, RF
环境中发现的许多药物和化学品要么被N-乙酰转移酶1(NAT1，EC 2.3.1.5)解毒并从体内消除，要么被激活为可能导致毒性和/或癌症的代谢物。NAT1在体内的活性受遗传多态和环境因素的调节，如底物依赖的下调和氧化应激。在这里，我们报道了突变的Nat1等位基因低蛋白表达导致慢乙酰化表型的分子机制，并表明类似的过程解释了Nat1底物对酶的下调。与野生型Nat1-4和活性等位酶Nat1-24相比，Nat1等位基因Nat1-14、Nat1-15、Nat1-17和Nat1-22缺乏酶活性，细胞内半衰期较短(接近4h)。失活的等位酶不能被辅因子乙酰化，导致26个S蛋白酶体的泛素化和快速降解。活性部位半胱氨酸68的定点突变证实了这一点。NAT1底物对氨基苯甲酸诱导通常稳定的NAT14泛素化，导致其快速降解。从这项研究中，我们得出结论，NAT1在细胞中要么以稳定的乙酰化状态存在，要么以不稳定的非乙酰化状态存在，并且阻止蛋白质乙酰化的Nat1基因突变会产生缓慢的乙酰化表型。
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 (similar to4 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.