Human N-acetyltransferase 2 (NAT2) is polymorphic in humans and may associate with cancer risk by modifying individual susceptibility to cancers from carcinogen exposure. Since molecular epidemiological studies investigating these associations usually include determining NAT2 single nucleotide polymorphisms (SNPs), haplotypes, or genotypes, their conclusions can be compromised by the uncertainty of genotype-phenotype relationships. We characterized NAT2 SNPs and haplotypes by cloning and expressing recombinant NAT2 allozymes in mammalian cells. The reference and variant recombinant NAT2 allozymes were characterized for arylamine N-acetylation and O-acetylation of N-hydroxy-arylamines. SNPs and haplotypes that conferred reduced enzymatic activity did so by reducing NAT2 protein without changing NAT2 mRNA levels. Among SNPs that reduced catalytic activity, G191A (R64Q), G590A (R197Q) and G857A (G286E) reduced protein half-life but T341C (I114T), G499A(E167K) and A411T (L137F) did not. G857A (G286E) and the major haplotype possessing this SNP (NAT2*7B) altered the affinity to both substrate and cofactor acetyl coenzyme A, resulting in reduced catalytic activity towards some substrates but not others. Our results suggest that coding region SNPs confer slow acetylator phenotype by multiple mechanisms that also may vary with arylamine exposures.
Cutaneous drug reactions (CDRs) associated with sulfonamides are believed to be mediated through the formation of reactive metabolites that result in cellular toxicity and protein haptenation. We evaluated the bioactivation and toxicity of sulfamethoxazole (SMX) and dapsone (DDS) in normal human dermal fibroblasts (NHDF). Incubation of cells with DDS or its metabolite (D-NOH) resulted in protein haptenation readily detected by confocal microscopy and ELISA. While the metabolite of SMX (S-NOH) haptenated intracellular proteins, adducts were not evident in incubations with SMX. Cells expressed abundant N-acetyltransferase-1 (NAT1) mRNA and activity, but little NAT2 mRNA or activity. Neither NAT1 nor NAT2 protein were detectable. Incubation of NHDF with S-NOH or D-NOH increased reactive oxygen species formation and reduced glutathione content. NHDF were less susceptible to the cytotoxic effect of S-NOH and D-NOH than are keratinocytes. Our studies provide the novel observation that NHDF are able to acetylate both arylamine compounds and bioactivate the sulfone, DDS, giving rise to haptenated proteins. The reactive metabolites of SMX and DDS also provoke oxidative stress in these cells in a time- and concentration-dependent fashion. Further work is needed to determine the role of the observed toxicity in mediating CDRs observed with these agents.
sulfonamides; cutaneous drug reactions; fibroblasts; protein haptenation; toxicity; N-acetyltransferase
Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine pro-carcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and highly polymorphic. While many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying the functional effects of specific NAT gene coding region SNPs. Analyses of mammalian NAT protein structures can greatly help to answer these questions. Homology modeling techniques can be used to approximate mammalian NAT structures using known bacterial NAT crystal structures as templates. In comparison to the bacterial template NATs used for homology modeling, mammalian NATs have a 17 residue insert of unknown structure and function. Homology modeling analyses yielded two different alignments (Modeller 8v1 or 3DCoffee algorithms) that placed this insert in two likely alternative locations. Secondary structure prediction techniques and experimental analyses of a series of human NAT2 mutants with artificial deletions/replacements of the insert region distinguished one of these alternatives as the most likely insert location and provided a better understanding of its structure and function. This study demonstrates both the utility and limitations of computational structural modeling with proteins that differ as much as the mammalian and bacterial NATs.
Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.
Human N-acetyltransferase 1 (NAT1) alleles are characterized by one or more single nucleotide polymorphisms (SNPs) associated with rapid and slow acetylation phenotypes. NAT1 both activates and deactivates arylamine drugs and carcinogens, and NAT1 polymorphisms are associated with increased frequencies of many cancers and birth defects. The recently resolved human NAT1 crystal structure was used to evaluate SNPs resulting in the protein substitutions R64W, V149I, R187Q, M205V, S214A, D251V, E261K, and I263V. The analysis enhances knowledge of NAT1 structure-function relationships, important for understanding associations of NAT1 SNPs with genetic predisposition to cancer, birth defects, and other diseases.
NAT1, N-acetyltransferase 1; single nucleotide polymorphisms (SNPs); structure-function relationships; cancer susceptibility; birth defects
Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) are important phase II enzymes involved in the biotransformation of xenobiotics. In toxicity and carcinogenicity studies, functional polymorphism of rat N-acetyltransferase is considered a model for similar human variability. To accurately quantitate expression of the three rat N-acetyltransferases, we developed sensitive, specific assays for Nat1, Nat2 and Nat3 mRNAs. In male F344 rats, tissue-specific expression varied over a limited range for both Nat1 (∼19-fold) and Nat2 (∼30-fold), with highest expression of both genes in colon. Nat3 mRNA was at least two to three orders of magnitude less than Nat1 or Nat2. Comparison of Nat1 and Nat2 mRNA expression in bladder, colon, liver and lung of male and female F344 rats detected no significant gender-specific difference. In Sprague Dawley and F344 rats ranging in age from neonate to mature adult, colon showed a >10-fold increase in Nat2 during the first postnatal month that did not correlate with changes in Nat1. In contrast, Nat2 showed no developmental change in Sprague Dawley or F344 liver as Nat1 increased modestly. These measures of rat Nat expression confirm that Nat3 expression is negligible and that Nat1 and Nat2 are the primary determinants of arylamine acetylation activity in all tested tissues. The findings demonstrate differential tissue-specific and developmental regulation of the rat Nat1 and Nat2 genes and contribute to more complete understanding of tissue-, gender-, and development-specific expression patterns of the cognate N-acetyltransferase genes of humans and other species.
N-acetyltransferases in humans (NAT1 and NAT2)
catalyse the acetylation of arylamines including food derived
heterocyclic arylamine carcinogens. Other substrates include the
sulphonamide 5-aminosalicylic acid (5-ASA), which is an NAT1 specific substrate; N-acetylation of 5-ASA is a major
route of metabolism. NAT1 and NAT2 are both polymorphic.
Aims—To investigate NAT expression in apparently
healthy human intestines in order to understand the possible role of
NAT in colorectal cancer and in the therapeutic response to 5-ASA.
Methods—The intestines of four organ donors were
divided into eight sections. DNA was prepared for genotyping
NAT1 and NAT2 and enzymic activities of NAT1
and NAT2 were determined in cytosols prepared from each
section. Tissue was fixed for immunohistochemistry with specific NAT
antibodies. Western blotting was carried out on all samples of cytosol
and on homogenates of separated muscle and villi after microdissection.
Results—NAT1 activity of all cytosols was greater
than NAT2 activity. NAT1 and NAT2 activities correlated with the
genotypes of NAT1 and NAT2 and with the levels
of NAT1 staining determined by western blotting. The ratio of NAT1:NAT2
activities showed interindividual variations from 2 to 70. NAT1
antigenic activity was greater in villi than in muscle. NAT1 was
detected along the length of the villi in the small intestine. In colon
samples there was less NAT1 at the base of the crypts with intense
staining at the tips.
Conclusions—The interindividual variation in NAT1
and NAT2 in the colon could affect how individuals respond to exposure
to specific NAT substrates including carcinogens and 5-ASA.
arylamine N-acetyltransferase; 5-aminosalicylate; colorectal cancer; drug metabolism; inflammatory
bowel disease; diet
Human N-acetyltransferase 2 (NAT2) genetic polymorphisms may modify drug efficacy and toxicity and individual cancer susceptibility from carcinogen exposure. A411T (L137F) and G364A (D122N) are two single nucleotide polymorphisms (SNPs) that coexist with other SNPs in human NAT2 alleles NAT2*5I and NAT2*12D, respectively. Cloning and expression in COS-1 cells showed that both A411T and G364A reduced NAT2 immunoreactive protein to an undetectable level without causing changes in mRNA level. Missense mutants displayed different effects on sulfamethazine N-acetylation activity for both L137 (wild-type: 70.2±5.2; L137F: 1.34±0.03; L137W: non-detectable; L137I: 34.2±2.0; L137G: 0.52±0.04 nmol/min/mg) and D122 (wildtype: 70.2±5.2; D122R: non-detectable; D122Q: non-detectable; D122E: 1.72±0.24 nmol/min/mg). To further test our hypothesis that A411T (L137F) and G364A (D122N) accelerate protein degradation, various NAT2 alleles were cloned and expressed in E. coli, which does not possess the ubiquitin-mediated degradation pathway. In contrast to the expression in mammalian cells, recombinant NAT2 possessing either of these two SNPs showed no reduction in immunoreactive NAT2 level when expressed in E. coli. These findings suggest that both A411T (L137F) and G364A (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D.
N-acetyltransferase-2; Single nucleotide polymorphism; protein degradation; slow acetylator phenotype
Acetylation by arylamine N-acetyltransferases (NATs) is a major route in the metabolism of numerous drugs and carcinogens. Recent studies suggest that the same enzymes also catalyze N,O-transacetylation and O-acetylation. A genetic polymorphism of clinical relevance divides the human population into slow and rapid acetylators of arylamines. Two human NATs, NAT1 and NAT2, have recently been characterized by protein purification, cloning, and functional expression of the respective genes; both were localized to chromosome 8. NAT1 codes for a protein with ubiquitous tissue distribution and a high affinity for p-aminobenzoic acid and p-aminosalicylic acid, so-called monomorphic substrates. NAT2 codes for a protein predominantly expressed in liver with a high affinity for sulfamethazine and other polymorphically metabolized drugs. NAT2 was analyzed at the level of protein, RNA and DNA derived from phenotyped slow and rapid acetylators. Two common (M1, M2) and one rare (M3) mutant allele were identified and their mutations characterized. A simple polymerase chain reaction-based DNA test can identify > 95% of mutant alleles and predict the phenotype.
Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10μM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype–dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP.
N-acetyltransferase 1; N-acetyltransferase 2; 4-aminobiphenyl; mammary epithelial cells; downregulation; heterocyclic amines
Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (PABA; selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene (AF) and 4-aminobiphenyl (ABP) N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (N-OH-PhIP) and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline] (N-OH-MeIQx) O-acetyltransferase activities were two to three-fold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 hr with 10 μM ABP showed down regulation in the N-and O-acetylation of all substrates tested except for the NAT1 selective substrate SMZ. This down regulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype dependent N- and O- acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to down regulation by the arylamine carcinogen 4-aminobiphenyl.
N-acetyltransferase 1; N-acetyltransferase 2; 4-aminobiphenyl; mammary epithelial cells; down regulation; heterocyclic amines
Aromatic amines such as 4-aminobiphenyl (ABP) require biotransformation to exert their carcinogenic effects. Genetic polymorphisms in biotransformation enzymes such as N-acetyltransferase 2 (NAT2) may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator), NAT2*5B (common Caucasian slow acetylator), or NAT2*7B (common Asian slow acetylator) alleles (haplotypes) were treated with ABP to test the effect of NAT2 polymorphisms on DNA adduct formation and mutagenesis. ABP N-acetyltransferase catalytic activities were detectable only in cell lines transfected with NAT2 and were highest in cells transfected with NAT2*4, lower in cells transfected with NAT2*7B, and lowest in cells transfected with NAT2*5B. Following ABP treatment, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the primary adduct formed. Cells transfected with both CYP1A1 and NAT2*4 showed the highest concentration-dependent cytotoxicity, hypoxanthine phosphoribosyl transferase (hprt) mutants, and dG-C8-ABP adducts. Cells transfected with CYP1A1 and NAT2*7B showed lower levels of cytotoxicity, hprt mutagenesis, and dG-C8-ABP adducts. Cells transfected with CYP1A1 only or cells transfected with both CYP1A1 and NAT2*5B did not induce cytotoxicity, hprt mutagenesis or dG-C8-ABP adducts. ABP DNA adduct levels correlated very highly (r > 0.96) with ABP-induced hprt mutant levels following each treatment. The results of the present study suggest that investigations of NAT2 genotype or phenotype associations with disease or toxicity could be more precise and reproducible if heterogeneity within the “slow” NAT2 acetylator phenotype is considered and incorporated into the study design.
N-acetyltransferase 2; Acetylator genotype; 4-aminobiphenyl; DNA adducts
Arylamine N-acetyltransferase 2 (NAT2) modifies drug efficacy/toxicity and cancer risk due to its role in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens. Human NAT2 alleles possess a combination of single nucleotide polymorphisms (SNPs) associated with slow acetylation phenotypes. Clinical and molecular epidemiology studies investigating associations of NAT2 genotype with drug efficacy/toxicity and/or cancer risk are compromised by incomplete and sometimes conflicting information regarding genotype/phenotype relationships. Studies in our laboratory and others have characterized the functional effects of SNPs alone, and in combinations present in alleles or haplotypes. We extrapolate this data generated following recombinant expression in yeast and COS-1 cells to assist in the interpretation of NAT2 structure. Whereas previous structural studies used homology models based on templates of N-acetyltransferase enzyme crystal structures from various prokaryotic species, alignment scores between bacterial and mammalian N-acetyltransferase protein sequences are low (~ 30%) with important differences between the bacterial and mammalian protein structures. Recently, the crystal structure of human NAT2 was released from the Protein Data Bank under accession number 2PFR. We utilized the NAT2 crystal structure to evaluate the functional effects of SNPs resulting in the protein substitutions R64Q (G191A), R64W (C190T), I114T (T341C), D122N (G364A), L137F (A411T), Q145P (A434C), E167K (G499A), R197Q (C590A), K268R (A803G), K282T (A845C), and G286E (G857A) of NAT2. This analysis advances understanding of NAT2 structure-function relationships, important for interpreting the role of NAT2 genetic polymorphisms in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens.
Human N-acetyltransferase 2 (NAT2); single nucleotide polymorphism (SNP); arylamine carcinogens; pharmacogenetics; cancer risk; structure/function
Naturally occurring antisense transcripts (NATs) are non-coding RNAs that may regulate the activity of sense transcripts to which they bind because of complementarity. NATs that are not located in the gene they regulate (trans-NATs) have better chances to evolve than cis-NATs, which is evident when the sense strand of the cis-NAT is part of a protein coding gene. However, the generation of a trans-NAT requires the formation of a relatively large region of complementarity to the gene it regulates.
Pseudogene formation may be one evolutionary mechanism that generates trans-NATs to the parental gene. For example, this could occur if the parental gene is regulated by a cis-NAT that is copied as a trans-NAT in the pseudogene. To support this we identified human pseudogenes with a trans-NAT to the parental gene in their antisense strand by analysis of the database of expressed sequence tags (ESTs). We found that the mutations that appeared in these trans-NATs after the pseudogene formation do not show the flat distribution that would be expected in a non functional transcript. Instead, we found higher similarity to the parental gene in a region nearby the 3' end of the trans-NATs.
Our results do not imply a functional relation of the trans-NAT arising from pseudogenes over their respective parental genes but add evidence for it and stress the importance of duplication mechanisms of genetic material in the generation of non-coding RNAs. We also provide a plausible explanation for the large transcripts that can be found in the antisense strand of some pseudogenes.
Genetic variants of human N-acetyltransferase 1 (NAT1) are associated with cancer and birth defects. N- and O-acetyltransferase catalytic activities, Michaelis-Menten kinetic constants (Km & Vmax), and steady state expression levels of NAT1-specific mRNA and protein were determined for the reference NAT1*4 and variant human NAT1 haplotypes possessing single nucleotide polymorphisms (SNPs) in the open reading frame. Although none of the SNPs caused a significant effect on steady state levels of NAT1-specific mRNA, C97T(R33stop), C190T(R64W), C559T (R187stop) and A752T(D251V) each reduced NAT1 protein level and/or N- and O-acetyltransferase catalytic activities to levels below detection. G560A(R187Q) substantially reduced NAT1 protein level and catalytic activities and increased substrate Km. The G445A(V149I), G459A(synonymous) and T640G(S214A) haplotype present in NAT1*11 significantly (p<0.05) increased NAT1 protein level and catalytic activity. Neither T21G(synonymous), T402C(synonymous), A613G(M205V), T777C(synonymous), G781A(E261K), or A787G(I263V) significantly affected Km, catalytic activity, mRNA or protein level. These results suggest heterogeneity among slow NAT1 acetylator phenotypes.
N-acetyltransferase 1; acetylator genotype; acetylator phenotype; single nucleotide polymorphisms (SNPs); functional SNPs; O-acetylation; N-acetylation; 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine
4,4'-Methylenedianiline (MDA) is widely used in the manufacture of polyurethane foam, epoxy resins and polymers. Exposure to MDA induces liver damage in humans and rats. MDA undergoes N-acetylation catalyzed by N-acetyltransferase 1 (NAT1) and 2 (NAT2) in the liver. Both human and rat NAT2 are polymorphic and human NAT2 genetic polymorphism modifies the frequency and/or severity of drug and xenobiotic toxicity in human populations. Recombinant expression of rat Nats in Escherichia coli showed that MDA was acetylated by both recombinant rat Nat1 and Nat2 and was catalyzed at substantially higher rates by rapid acetylator Nat2 compared to slow acetylator Nat2. Rapid acetylator F344 rat liver cytosols catalyzed the N-acetylation of MDA at significantly higher rates than those from slow acetylator WKY inbred rats. To test the effect of NAT2 genetic polymorphism on hepatotoxicity from acute MDA exposure, we compared hepatotoxicity in rapid (F344) and slow (WKY) Nat2 acetylator inbred rats administered MDA. Based on the results of dose-response studies ranging up to 150 mg/kg of MDA administered by intragastric gavage, the effect of a moderately hepatotoxic dose (37.5 mg/kg) was compared in rapid vs. slow acetylator rats. Plasma alanine transaminase (ALT) enzyme activities were approximately 5-fold higher (p < 0.05) in rapid vs. slow acetylator rats after MDA treatment and necrotizing hepatitis with portal damage consisting of bile ductular necrosis, portal expansion and inflammation was clearly more prominent. These results suggest that acetylator phenotype is an important factor for susceptibility towards MDA hepatotoxicity.
Protein Nα-terminal acetylation (Nt-acetylation) is considered one of the most common protein modification in eukaryotes, and 80-90% of all soluble human proteins are modified in this way, with functional implications ranging from altered protein function and stability to translocation potency amongst others. Nt-acetylation is catalyzed by N-terminal acetyltransferases (NATs), and in yeast five NAT types are identified and denoted NatA-NatE. Higher eukaryotes additionally express NatF. Except for NatD, human orthologues for all yeast NATs are identified. yNatD is defined as the catalytic unit Naa40p (Nat4) which co-translationally Nt-acetylates histones H2A and H4.
In this study we identified and characterized hNaa40p/hNatD, the human orthologue of the yeast Naa40p. An in vitro proteome-derived peptide library Nt-acetylation assay indicated that recombinant hNaa40p acetylates N-termini starting with the consensus sequence Ser-Gly-Gly-Gly-Lys-, strongly resembling the N-termini of the human histones H2A and H4. This was confirmed as recombinant hNaa40p Nt-acetylated the oligopeptides derived from the N-termini of both histones. In contrast, a synthetically Nt-acetylated H4 N-terminal peptide with all lysines being non-acetylated, was not significantly acetylated by hNaa40p, indicating that hNaa40p catalyzed H4 Nα-acetylation and not H4 lysine Nε-acetylation. Also, immunoprecipitated hNaa40p specifically Nt-acetylated H4 in vitro. Heterologous expression of hNaa40p in a yeast naa40-Δ strain restored Nt-acetylation of yeast histone H4, but not H2A in vivo, probably reflecting the fact that the N-terminal sequences of human H2A and H4 are highly similar to each other and to yeast H4 while the N-terminal sequence of yeast H2A differs. Thus, Naa40p seems to have co-evolved with the human H2A sequence. Finally, a partial co-sedimentation with ribosomes indicates that hNaa40p co-translationally acetylates H2A and H4. Combined, our results strongly suggest that human Naa40p/NatD is conserved from yeast. Thus, the NATs of all classes of N-terminally acetylated proteins in humans now appear to be accounted for.
N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.
Small chemical groups are commonly attached to proteins in order to control their activity, localization, and stability. An abundant protein modification is N-terminal acetylation, in which an N-terminal acetyltransferase (NAT) catalyzes the transfer of an acetyl group to the very N-terminal amino acid of the protein. When going from lower to higher eukaryotes there is a significant increase in the occurrence of N-terminal acetylation. We demonstrate here that this is partly because higher eukaryotes uniquely express NatF, an enzyme capable of acetylating a large group of protein N-termini including those previously found to display an increased N-acetylation potential in higher eukaryotes. Thus, the current study has possibly identified the last major component of the eukaryotic machinery responsible for co-translational N-acetylation of proteins. All eukaryotic proteins start with methionine, which is co-translationally cleaved when the second amino acid is small. Thereafter, NatA may acetylate these newly exposed N-termini. Interestingly, NatF also has the potential to act on these types of N-termini where the methionine was not cleaved. At the cellular level, we further found that NatF is essential for normal chromosome segregation during cell division.
The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast Nα-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the Nα-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for Nα-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Δnat1 or Δard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Δnat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Δnat1 or Δard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.
Animal studies suggest that lymphomagenesis can be induced by exposure to carcinogenic aromatic and heterocyclic amines found in diet, cigarette smoke, and the environment, but human epidemiologic investigations of these exogenous exposures have yielded conflicting results. As part of our evaluation of the role of aromatic and heterocyclic amines, which are metabolized by N-acetyltransferase (NAT) enzymes, in the etiology of non-Hodgkin lymphoma (NHL), we examined NHL risk in relation to genetic variation in NAT1 and NAT2 and exposure to cigarette smoke and dietary heterocyclic amines and mutagens.
We genotyped ten common single nucleotide polymorphisms (SNPs) in NAT1 and NAT2 among 1136 cases and 922 controls from a population-based case–control study in four geographic areas of the US. Relative risk of NHL for NAT1 and NAT2 genotypes, NAT2 acetylation phenotype, and exposure to cigarette smoke and dietary heterocyclic amines and mutagens was estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from unconditional logistic regression models.
We observed increased risk of NHL among individuals with the NAT1*10/*10 genotype compared with individuals with other NAT1 genotypes (OR=1.60, 95% CI 1.04–2.46, p=0.03). We also observed increased NHL risk in a dose-dependent model among NAT2 intermediate- and rapid-acetylators in comparison with slow-acetylators, although only the trend was statistically significant (intermediate: OR=1.18, 95% CI 0.97–1.44, p=0.1; rapid: OR=1.43, 95% CI 0.97–2.14, p=0.07; p for linear trend=0.03). Compared with nonsmokers, NHL risk estimates for current cigarette smoking were increased only among NAT2 intermediate/rapid-acetylators (OR=2.44, 95% CI 1.15–5.20, p=0.02).
Our data provide evidence that NAT1 and NAT2 genotypes are associated with NHL risk and support a contributory role for carcinogenic aromatic and/or heterocyclic amines in the multi-factorial etiology of NHL.
lymphoma, non-Hodgkin; N-acetyltransferase 1; N-acetyltransferase 2; genetic variation; polymorphism, single nucleotide
Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.
Arylamine N-acetyltransferase/NAT; adrenal; embryo; breast cancer
Arylamine N-acetyltransferases (NAT; EC 18.104.22.168) catalyze both the N-acetylation and O-acetylation of arylamines and hydrazines. Humans possess two functional N-acetyltransferase genes, NAT1 and NAT2, as well as a non-functional pseudogene, NATP. Previous studies have identified Nat1 and Nat2 genes in the rat. In this study, we identified and characterized a third rat N-acetyltransferase gene (Nat3) consisting of a single open reading frame of 870 base pairs encoding a 290 amino acid protein, analogous to the previously identified human and rat N-acetyltransferase genes. Rat Nat3 nucleotide sequence was 77.2% and 75.9% identical to human NAT1 and NAT2, respectively. Rat Nat3 amino acid sequence was 68.6% and 67.2% identical to human NAT1 and NAT2, respectively. Rat Nat1, Nat2, and Nat3 were each cloned and recombinantly expressed in Escherichia coli. Recombinant rat Nat3 exhibited thermostability intermediate between recombinant rat Nat1 and Nat2. Recombinant rat Nat3 was functional and catalyzed the N-acetylation of several arylamine substrates, including 3-ethylaniline, 3,5-dimethylaniline, 5-aminosalicylic acid, 4-aminobiphenyl, 4,4′-methylenedianiline, 4,4′-methylenebis(2-chloroaniline), and 2-aminofluorene, and the O-acetylation of N-hydroxy-4-aminobiphenyl. The relative affinities of arylamine carcinogens such as 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and 2-aminofluorene for N-and O-acetylation via recombinant rat Nat3 were comparable to recombinant rat Nat1 and higher than for recombinant rat Nat2. This study is the first to report a third arylamine N-acetyltransferase isozyme with significant functional capacity.
The sulfamethoxazole (SMX)-trimethoprim drug combination is routinely used as prophylaxis against Pneumocystis pneumonia during the first 3 to 6 months after renal transplantation. The objective of this study was to examine the impact of N-acetyltransferase 2 (NAT2) and CYP2C9 polymorphisms on the pharmacokinetics of SMX in 118 renal transplant recipients. Starting on day 14 after renal transplantation, patients were administered 400 mg/day-80 mg/day of SMX-trimethoprim orally once daily. On day 14 after the beginning of SMX therapy, plasma SMX concentrations were determined by a high-performance liquid chromatography method. The SMX area under the concentration-time curve from 0 to 24 h (AUC0-24) for 15 recipients with the NAT2 slow acetylator genotype (NAT2*5/*6, -*6/*6, -*6/*7, and -*7/*7) was significantly greater than that for 56 recipients with the NAT2 rapid acetylator genotype (homozygous for NAT2*4) (766.4 ± 432.3 versus 537.2 ± 257.5 μg-h/ml, respectively; P = 0.0430), whereas there were no significant differences in the SMX AUC0-24 between the CYP2C9*1/*1 and -*1/*3 groups. In a multiple regression analysis, the SMX AUC0-24 was associated with NAT2 slow acetylator polymorphisms (P = 0.0095) and with creatinine clearance (P = 0.0499). Hepatic dysfunction in NAT2 slow acetylator recipient patients during the 6-month period after SMX administration was not observed. SMX plasma concentrations were affected by NAT2 polymorphisms and renal dysfunction. Although standard SMX administration to patients with NAT2 slow acetylator polymorphisms should be accompanied by monitoring for side effects and drug interaction effects from the inhibition of CYP2C9, SMX administration at a low dose (400 mg) as prophylaxis may not provide drug concentrations that reach the level necessary for the expression of side effects. Further studies with a larger sample size should be able to clarify the relationship between SMX plasma concentration and side effects.
Genetic variations in xenobiotic metabolizing genes can influence susceptibility to many environmentally induced cancers. Inheritance of the N-acetyltransferase 1 allele (NAT1*10), linked with increased metabolic activation of pro-carcinogens, is associated with an increased susceptibility to many cancers in which cigarette- or meat-derived carcinogens have been implicated in their etiology. The role of NAT1*10 in prostate cancer is under studied. Although cigarette smoking is not considered a risk factor for prostate cancer, a recent review suggests it may play a role in disease progression. Consequently, we examined the association of NAT1*10 with prostate cancer risk, grade, and stage among 400 Finnish male smokers using a case–control study design. Following genotyping of 206 patients and 196 healthy controls, our results do not support the role of NAT1*10 in relation to prostate cancer risk (OR = 1.28; 95% CI, 0.66–2.47), aggressive disease (OR = 0.58; 95% CI, 0.13–2.67), or advanced disease (OR = 1.19; 95% CI, 0.49–2.91).
N-acetyltransferase 1; Prostate cancer; Disease progression; Arylamine carcinogens
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in multiple organs and numerous species. Bioactivation of PhIP is initiated by PhIP N2- hydroxylation catalyzed by cytochrome P450s. Following N-hydroxylation, O-acetylation catalyzed by N-acetyltransferase 2 (NAT2) is considered a further possible activation pathway. Genetic polymorphisms in NAT2 may modify cancer risk following exposure.Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were used to test the effect of CYP1A1 and NAT2 polymorphism on PhIP genotoxicity.Cells transfected with NAT2*4 had significantly higher levels of N-hydroxy-PhIP O-acetyltransferase (P = 0.0150) activity than cells transfected with NAT2*5B. Following PhIP treatment, CHO cell lines transfected with CYP1A1, CYP1A1/NAT2*4 and CYP1A1/NAT2*5B each showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis not observed in untransfected CHO cells.dG-C8-PhIP was the primary DNA adduct formed and levels were dose-dependent in transfected CHO cells in the order: CYP1A1 < CYP1A1 & NAT2*5B < CYP1A1 & NAT2*4 although levels did not differ significantly (P>0.05) following one-way analysis of variance.These results strongly support activation of PhIP by CYP1A1 with little effect of human NAT2 genetic polymorphism on mutagenesis and DNA damage.
PhIP; 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; CYP1A1; cytochrome P4501A1; NAT2; N-acetyltransferase 2