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Phenotype of the most common "slow acetylator" arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent.

ABSTRACT: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II cytosolic enzyme responsible for the activation or deactivation of many arylamine compounds including pharmaceuticals and environmental carcinogens. NAT1 is highly polymorphic and has been associated with altered risk toward many cancers. NAT1*14B is characterized by a single nucleotide polymorphism in the coding region (rs4986782; 560G>A; R187Q). NAT1*14B is associated with higher frequency of smoking-induced lung cancer and is the most common "slow acetylator" arylamine NAT1 genetic variant. Previous studies have reported decreased N- and O-acetylation capacity and increased proteasomal degradation of NAT1 14B compared with the referent, NAT1 4. The current study is the first to investigate NAT1*14B expression using constructs that completely mimic NAT1 mRNA by including the 5'- and 3'-untranslated regions, together with the open reading frame of the referent, NAT1*4, or variant, NAT1*14B. Our results show that NAT1 14B is not simply associated with "slow acetylation." NAT1 14B-catalyzed acetylation phenotype is substrate-dependent, and NAT1 14B exhibits higher N- and O-acetylation catalytic efficiency as well as DNA adducts after exposure to the human carcinogen 4-aminobiphenyl.

SUBMITTER: Millner LM

PROVIDER: S-EPMC3250052 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Phenotype of the most common "slow acetylator" arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent.

Millner Lori M LM Doll Mark A MA Cai Jian J States J Christopher JC Hein David W DW

Drug metabolism and disposition: the biological fate of chemicals 20111018 1

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II cytosolic enzyme responsible for the activation or deactivation of many arylamine compounds including pharmaceuticals and environmental carcinogens. NAT1 is highly polymorphic and has been associated with altered risk toward many cancers. NAT1*14B is characterized by a single nucleotide polymorphism in the coding region (rs4986782; 560G>A; R187Q). NAT1*14B is associated with higher frequency of smoking-induced lung cancer and is the most ...[more]

PMID: 22010219

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Project description:The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

Project description:BackgroundThere are many molecular differences between estrogen receptor α (ERα)-positive and ER-negative breast cancers. Recent analyses have shown that the former can be divided into two subtypes, luminal A and luminal B. These differ in response to endocrine therapy and chemotherapy, and in prognosis. In a previous study, we found that microRNA (miR)-1290 that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1). The aim of the present study was to determine whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis.MethodsLuciferase reporter assays were employed to validate NAT1 as a putative miR-1290 target gene. Expression of NAT1, ERα, progesterone receptor (PgR) and HER2 was analyzed in 394 breast cancer samples by immunohistochemistry.ResultsNAT1 was confirmed to be a direct target of miR-1290. Levels of expression of NAT1 were positively correlated with those of ERα (P < 0.0001) and PgR (P < 0.0001), but negatively correlated with both tumor grade and size (P < 0.0001). Kaplan-Meier analysis showed that the presence of NAT1 was significantly associated with increased overall survival (OS) (P = 0.0416) in these patients. Similarly, significant associations of NAT1 with disease-free survival (DFS) (P = 0.0048) and OS (P = 0.0055) in those patients who received adjuvant endocrine therapy with tamoxifen (n = 176) were found. Moreover, NAT1 was also significantly associated with increased DFS (P = 0.0025) and OS (P = 0.0007) in the subset of lymph node-positive patients (n = 147). Univariate and multivariate analyses showed significant associations between levels of NAT1 and DFS (P = 0.0005 and 0.019, respectively).ConclusionsWe report that miR-1290 directly targets the NAT1 3'-UTR and that NAT1 protein expression is correlated with improved OS of breast cancer patients. NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its target NAT1 are associated with important characteristics of breast cancer.

Project description:Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

Dataset Information

Phenotype of the most common "slow acetylator" arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent.

Publications

Phenotype of the most common "slow acetylator" arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent.

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