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1.
J Clin Invest ; 125(4): 1739-51, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25798622

ABSTRACT

Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.


Subject(s)
Arylamine N-Acetyltransferase/physiology , Insulin Resistance/physiology , Mutation, Missense , Point Mutation , 3T3-L1 Cells , Adipogenesis/drug effects , Adipogenesis/physiology , Adolescent , Adult , Animals , Arylamine N-Acetyltransferase/deficiency , Arylamine N-Acetyltransferase/genetics , Asian People/genetics , Child , Coronary Disease/enzymology , Coronary Disease/genetics , Europe/epidemiology , Female , Gene Frequency , Genome-Wide Association Study , Glucose/metabolism , Glycated Hemoglobin/analysis , Hispanic or Latino/genetics , Humans , Hyperglycemia/enzymology , Hyperglycemia/genetics , Hypertriglyceridemia/enzymology , Hypertriglyceridemia/genetics , Isoenzymes/deficiency , Isoenzymes/physiology , Lipolysis/drug effects , Lipolysis/physiology , Male , Mice , Mice, Knockout , Middle Aged , Polymorphism, Single Nucleotide , Prospective Studies , Taiwan/epidemiology , United States/epidemiology , White People/genetics , Young Adult
2.
Cancer Med ; 4(4): 565-74, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25627111

ABSTRACT

Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and ß-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers.


Subject(s)
Arylamine N-Acetyltransferase/physiology , Isoenzymes/physiology , Triple Negative Breast Neoplasms/enzymology , Animals , Arylamine N-Acetyltransferase/deficiency , Cadherins/metabolism , Cell Line, Tumor , Female , Gene Knockdown Techniques , Humans , Isoenzymes/deficiency , Lentivirus , Lung Neoplasms/secondary , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Pseudopodia/physiology , Snail Family Transcription Factors , Transcription Factors/metabolism , Transduction, Genetic , Triple Negative Breast Neoplasms/pathology , Tumor Cells, Cultured , beta Catenin/metabolism
3.
PLoS One ; 8(10): e77923, 2013.
Article in English | MEDLINE | ID: mdl-24205029

ABSTRACT

Folate catabolism involves cleavage of the C(9)-N(10) bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.


Subject(s)
Arylamine N-Acetyltransferase/physiology , Folic Acid/metabolism , Glutamates/metabolism , S-Adenosylmethionine/metabolism , Tetrahydrofolates/metabolism , Acetylation , Animals , Female , Folic Acid/analogs & derivatives , Humans , Kidney/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Sequence Deletion , Sex Factors
4.
PLoS One ; 7(8): e42797, 2012.
Article in English | MEDLINE | ID: mdl-22905173

ABSTRACT

BACKGROUND: There have been an increasing number of studies with evidence suggesting that the N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) genotypes may be implicated in the development of colorectal cancer (CRC) and colorectal adenoma (CRA). So far the published data on this association has remained controversial, however. We performed a meta-analysis of case-cohort and case-control studies using a subset of the published data, with an aim to derive a better understanding of the underlying relationship. METHODS/PRINCIPAL FINDINGS: A literature search was performed using Medline database for relevant studies published through October 31, 2011. A total of 39 publications were selected for this meta-analysis, including 11,724 cases and 16,215 controls for CRC, and 3,701 cases and 5,149 controls for CRA. In our pooled analysis of all these studies, the results of our meta-analysis suggested that the NAT1 genotype was not significantly associated with an elevated CRC risk (OR 0.99, 95% CI 0.91-1.07). We also found that individuals with the rapid NAT2 genotype did have an elevated risk of CRC (OR 1.07, 95% CI 1.01-1.13). There was no evidence for an association between the NAT1 and 2 rapid genotype and an elevated CRA risk (NAT1: OR 1.14, 95% CI 0.99-1.29; NAT2: OR 0.94, 95% CI 0.86-1.03). CONCLUSION: This meta-analysis suggests that individuals with NAT2 genotype had an elevated risk of CRC. There was no evidence for the association between NAT1 and 2 rapid genotype and CRA risk.


Subject(s)
Acetyltransferases/genetics , Adenoma/genetics , Arylamine N-Acetyltransferase/genetics , Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Isoenzymes/genetics , Polymorphism, Genetic , Arylamine N-Acetyltransferase/physiology , Case-Control Studies , Cohort Studies , Genetic Predisposition to Disease , Genetic Variation , Genotype , Humans , Isoenzymes/physiology , Risk
5.
Pharmacol Rev ; 64(1): 147-65, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22090474

ABSTRACT

The human arylamine N-acetyltransferases first attracted attention because of their role in drug metabolism. However, much of the current literature has focused on their role in the activation and detoxification of environmental carcinogens and how genetic polymorphisms in the genes create predispositions to increased or decreased cancer risk. There are two closely related genes on chromosome 8 that encode the two human arylamine N-acetyltransferases--NAT1 and NAT2. Although NAT2 has restricted tissue expression, NAT1 is found in almost all tissues of the body. There are several single-nucleotide polymorphisms in the protein coding and 3'-untranslated regions of the gene that affect enzyme activity. However, NAT1 is also regulated by post-translational and environmental factors, which may be of greater importance than genotype in determining tissue NAT1 activities. Recent studies have suggested a novel role for this enzyme in cancer cell growth. NAT1 is up-regulated in several cancer types, and overexpression can lead to increased survival and resistance to chemotherapy. Although a link to folate homeostasis has been suggested, many of the effects attributed to NAT1 and cancer cell growth remain to be explained. Nevertheless, the enzyme has emerged as a viable candidate for drug development, which should lead to small molecule inhibitors for preclinical and clinical evaluation.


Subject(s)
Arylamine N-Acetyltransferase , Drug Discovery , Isoenzymes , Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Arylamine N-Acetyltransferase/antagonists & inhibitors , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/physiology , Down-Regulation , Epigenesis, Genetic , Folic Acid/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/physiology , Molecular Structure , Neoplasms/enzymology , Protein Conformation , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Substrate Specificity
6.
Pharmacogenomics ; 12(8): 1091-3, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21843062

ABSTRACT

Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolizing enzymes playing a key role in the detoxification and metabolic activation of aromatic amine xenobiotics. The triennial International NAT Workshop has been an important academic meeting where developments in the study of NATs and aromatic amine metabolism have been presented. The 2010 Workshop took place in University Paris Diderot Paris, France. Topics included: structures and functions of eukaryotic and prokaryotic NATs, gene regulation and expression of human NATs, polymorphisms and their effects, arylamine metabolism and toxicity. Nomenclature issues were also discussed.


Subject(s)
Arylamine N-Acetyltransferase/genetics , Amino Acids, Aromatic/metabolism , Animals , Arylamine N-Acetyltransferase/metabolism , Arylamine N-Acetyltransferase/physiology , Bacteria/enzymology , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Enzymologic/physiology , Humans , Polymorphism, Genetic/genetics , Xenobiotics/metabolism
7.
Life Sci ; 86(3-4): 103-6, 2010 Jan 16.
Article in English | MEDLINE | ID: mdl-19932120

ABSTRACT

AIMS: To determine whether increased N-acetyltransferase (NAT) activity might have a toxic effect during development and an influence on folate levels since previous work has shown that only low levels of exogenous NAT can be achieved in constitutionally transgenic mice (Cao et al. 2005). MAIN METHODS: A human NAT1 tet-inducible construct was used that would not be expressed until the inducer was delivered. Human NAT1 cDNA was cloned into pTRE2 and injected into mouse oocytes. Two transgenic lines were crossed to mouse line TgN(rtTahCMV)4Uh containing the CMV promoted "tet(on)". Measurements of red blood cell folate levels in inbred strains of mice were performed. KEY FINDINGS: Only low levels of human NAT1 could be achieved in kidney (highly responsive in other studies) whether the inducer, doxycycline, was given by gavage or in drinking water. An inverse correlation of folate levels with Nat2 enzyme activity was found. SIGNIFICANCE: Since increasing NAT1 activity decreases folate in at least one tissue, the detrimental effect of expression of human NAT1 in combination with endogenous mouse Nat2 may be a consequence of increased catabolism of folate.


Subject(s)
Arylamine N-Acetyltransferase/metabolism , Folic Acid/blood , Acetylation , Animals , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/physiology , Cloning, Molecular , Cytomegalovirus/genetics , Doxycycline/pharmacology , Erythrocytes/metabolism , Female , Folic Acid/metabolism , Genes, Reporter , Genetic Vectors , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Isoenzymes/physiology , Kidney/enzymology , Liver/enzymology , Male , Mice , Mice, Transgenic , Species Specificity , Transgenes
8.
BMC Biochem ; 10: 15, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19480662

ABSTRACT

BACKGROUND: Protein acetylation is among the most common protein modifications. The two major types are post-translational Nepsilon-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational Nalpha-terminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (N alpha acetyltransferase 10 protein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val- and Cys- N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini in vitro and in vivo. RESULTS: We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of hNAA15 and hNAA16. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini in vitro. hNaa16p is ribosome associated, supporting its potential role in cotranslational Nalpha-terminal acetylation. hNAA16 is expressed in a variety of human cell lines, but is generally less abundant as compared to hNAA15. Specific knockdown of hNAA16 induces cell death, suggesting an essential role for hNaa16p in human cells. CONCLUSION: At least two distinct NatA protein Nalpha-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for Nalpha-terminal acetylation as compared to lower eukaryotes.


Subject(s)
Acetyltransferases/metabolism , Arylamine N-Acetyltransferase/physiology , Multienzyme Complexes/physiology , Acetylation , Amino Acid Sequence , Arylamine N-Acetyltransferase/metabolism , Cell Death , Cell Line, Tumor , Evolution, Molecular , Humans , Isoenzymes/metabolism , Molecular Sequence Data , N-Terminal Acetyltransferase E , N-Terminal Acetyltransferases , Peptides/metabolism , Phylogeny , Protein Processing, Post-Translational , Ribosomes/metabolism , Sequence Alignment
9.
Expert Opin Drug Metab Toxicol ; 5(4): 353-66, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19379125

ABSTRACT

Arylamine N-acetyltransferase 1 and 2 exhibit single nucleotide polymorphisms in human populations that modify drug and carcinogen metabolism. This paper updates the identity, location and functional effects of these single nucleotide polymorphisms and then follows with emerging concepts for understanding why pharmacogenetic findings may not be replicated consistently. Using this paradigm as an example, laboratory-based mechanistic analyses can reveal complexities such that genetic polymorphisms become biologically and medically relevant when confounding factors are more fully understood and considered. As medical care moves to a more personalized approach, the implications of these confounding factors will be important in understanding the complexities of personalized medicine.


Subject(s)
Arylamine N-Acetyltransferase/genetics , Isoenzymes/genetics , Pharmacogenetics/trends , Polymorphism, Single Nucleotide/genetics , Precision Medicine , Animals , Arylamine N-Acetyltransferase/chemistry , Arylamine N-Acetyltransferase/physiology , Humans , Isoenzymes/chemistry , Isoenzymes/physiology , Pharmacogenetics/methods
11.
Pharmacogenomics ; 9(6): 765-71, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18518853

ABSTRACT

Arylamine N-acetyltransferase (NAT) research has been influenced in recent years by the rapid progress in genomics, proteomics, structural genomics and other cutting-edge disciplines. To keep up with these advancements, the NAT scientific community has fostered collaboration and exchange of know-how between its members. As a specialized event bringing together experts from many different laboratories, the triennial International NAT Workshop has been instrumental in maintaining this culture over the past ten years. The 2007 Workshop took place in Alexandroupolis, Greece, and covered ongoing research on the structure and enzymatic function of human NATs, the prokaryotic and eukaryotic models for NAT, the mechanisms of NAT gene regulation and expression, the frequencies and effects of polymorphisms in the human NAT genes, and the involvement of NATs in multifactorial diseases, including cancer, allergic conditions, endometriosis and endemic nephropathies. Gene nomenclature issues were also addressed and the participants discussed current trends in the field.


Subject(s)
Arylamine N-Acetyltransferase , Biomedical Research/trends , Animals , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/metabolism , Arylamine N-Acetyltransferase/physiology , Gene Expression Regulation, Enzymologic , Humans , Models, Molecular , Species Specificity
12.
Eur J Hum Genet ; 16(2): 243-51, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18043717

ABSTRACT

The arylamine N-acetyltransferase 2 (NAT2) enzyme detoxifies a wide spectrum of naturally occurring xenobiotics including carcinogens and drugs. Variation at the NAT2 gene has been linked to the human acetylation capacity, either 'slow' or 'fast', which modifies susceptibility to cancer and adverse drug reactions. We investigated the possible influence of natural selection in shaping the acetylation phenotype and the NAT2 gene variability in six Central Asian populations, who are either long-term sedentary agriculturalists (two Tajik populations), recent sedentary agriculturalists (Kazakhs, Uzbeks) or nomad pastoralists (two Kirghiz populations). To this end, we sequenced the entire NAT2 coding exon, as well as genotyping nine intergenic SNPs covering a 200-kb region. Our results revealed that the two Tajik populations exhibited significantly higher proportions of slow acetylators than the nomadic populations. In addition, sequence-based neutrality tests yielded significantly positive values in Central Asian populations following an agriculturalist lifestyle, due to an excess of haplotypes at intermediate frequencies. Taken together, our data suggest that balancing selection, and/or directional selection on standing low-frequency alleles, have shaped NAT2 genetic diversity and the human acetylation phenotype in Central Asian agriculturalists. These results further support the hypothesis that a major transition in human lifestyle, such as the emergence of farming has dramatically changed human chemical environments and the selective pressures they imposed.


Subject(s)
Adaptation, Physiological/genetics , Agriculture , Arylamine N-Acetyltransferase/genetics , Genetic Variation , Acetylation , Arylamine N-Acetyltransferase/physiology , Asia, Central , Genetics, Population , Humans , Linkage Disequilibrium , Mutation , Phenotype , Polymorphism, Single Nucleotide
13.
Chem Biodivers ; 4(8): 1842-57, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17712822

ABSTRACT

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.


Subject(s)
Arachidonic Acids/biosynthesis , Arachidonic Acids/chemistry , Arylamine N-Acetyltransferase/physiology , Cannabinoid Receptor Modulators/chemistry , Endocannabinoids , Isoenzymes/physiology , Phospholipase D/physiology , Polyunsaturated Alkamides/chemistry , Animals , Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/metabolism , Humans , Polyunsaturated Alkamides/metabolism
14.
J Med Genet ; 44(9): 570-8, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17513527

ABSTRACT

BACKGROUND: Age-related hearing impairment (ARHI) is the most common sensory impairment in older people, affecting 50% of those aged 80 years. The proportion of older people is increasing in the general population, and as a consequence, the number of people affected with ARHI is growing. ARHI is a complex disorder, with both environmental and genetic factors contributing to the disease. The first studies to elucidate these genetic factors were recently performed, resulting in the identification of the first two susceptibility genes for ARHI, NAT2 and KCNQ4. METHODS: In the present study, the association between ARHI and polymorphisms in genes that contribute to the defence against reactive oxygen species, including GSTT1, GSTM1 and NAT2, was tested. Samples originated from seven different countries and were combined into two test population samples, the general European population and the Finnish population. Two distinct phenotypes for ARHI were studied, Z(low) and Z(high), representing hearing in the low and high frequencies, respectively. Statistical analysis was performed for single polymorphisms (GSTM1, GSTT1, NAT2*5A, NAT2*6A, and NAT2*7A), haplotypes, and gene-environment and gene-gene interactions. RESULTS: We found an association between ARHI and GSTT1 and GSTM1 in the Finnish population sample, and with NAT2*6A in the general European population sample. The latter finding replicates previously published data. CONCLUSION: As replication is considered the ultimate proof of true associations in the study of complex disorders, this study provides further support for the involvement of NAT2*6A in ARHI.


Subject(s)
Arylamine N-Acetyltransferase/genetics , Hearing Disorders/genetics , Polymorphism, Single Nucleotide , Age of Onset , Aged , Arylamine N-Acetyltransferase/physiology , Environment , Epistasis, Genetic , Europe/epidemiology , Female , Finland/epidemiology , Gene Frequency , Glutathione Transferase/genetics , Glutathione Transferase/physiology , Haplotypes/genetics , Hearing Disorders/epidemiology , Hearing Loss, High-Frequency/epidemiology , Hearing Loss, High-Frequency/genetics , Humans , Male , Middle Aged , Oxidative Stress/genetics
15.
Carcinogenesis ; 28(8): 1665-71, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17434923

ABSTRACT

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 toward 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.


Subject(s)
Arylamine N-Acetyltransferase/genetics , Haplotypes , Polymorphism, Single Nucleotide , Acetylation , Animals , Arylamine N-Acetyltransferase/metabolism , Arylamine N-Acetyltransferase/physiology , COS Cells , Chlorocebus aethiops , Humans , Phenotype , Sulfamethazine/antagonists & inhibitors , Sulfamethazine/metabolism
16.
J Bacteriol ; 189(5): 2155-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17158669

ABSTRACT

An arylamine N-acetyltransferase (NAT) responsible for the N acetylation of exogenous 3-amino-4-hydroxybenzoic acid in Streptomyces griseus was identified and characterized. This enzyme was distinct from other eukaryotic and bacterial NATs in that it acetylated various 2-aminophenol derivatives more effectively than it acetylated 5-aminosalicylic acid, and thus it may be involved in the metabolism of xenobiotic compounds.


Subject(s)
Aminobenzoates/metabolism , Aminophenols/metabolism , Arylamine N-Acetyltransferase/physiology , Streptomyces griseus/metabolism , Acetylation , Arylamine N-Acetyltransferase/genetics , Base Sequence , Escherichia coli/genetics , Hydroxybenzoates , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Substrate Specificity
17.
Cancer Res ; 66(21): 10541-7, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17079477

ABSTRACT

Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.


Subject(s)
Carcinogens/metabolism , Colon/metabolism , DNA Adducts/urine , Imidazoles/metabolism , Arylamine N-Acetyltransferase/physiology , Arylsulfotransferase/physiology , Cytochrome P-450 CYP1A2/physiology , Glucuronosyltransferase/physiology , Humans
18.
J Mol Biol ; 361(3): 482-92, 2006 Aug 18.
Article in English | MEDLINE | ID: mdl-16857211

ABSTRACT

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.


Subject(s)
Arylamine N-Acetyltransferase/physiology , Isoenzymes/physiology , Polymorphism, Genetic , Ubiquitin/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/metabolism , Catalysis , Cloning, Molecular , Cricetinae , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , HeLa Cells , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
19.
Asian Pac J Cancer Prev ; 7(2): 267-73, 2006.
Article in English | MEDLINE | ID: mdl-16839221

ABSTRACT

Tobacco smoking is an unequivocal risk factor for cancers of the larynx, lung, pancreas and urinary bladder. Whereas African-Americans demonstrate higher laryngeal, lung and pancreatic cancer rates than their Caucasian-American counterparts, they paradoxically have only approximately half of the urinary bladder incidences. One possible explanation is their N-acetyltransferase (NAT) status, since this enzyme is responsible for metabolism of arylamines in smoke and blacks are reported to have a higher rate for rapid acetylation than whites. However, other tobacco-related cancers are also linked to slow acetylation so that African-Americans might therefore also be expected to have lower incidences of other tobacco-related cancers. The present investigation was conducted with data from Cancer Incidence in Five Continents Vol VIII to assess whether there might be correlations between incidence rates for four major cancers across registries in the United States. Cluster analysis demonstrated clear separation of the white and black populations for all states, and significant correlations were observed between bladder and laryngeal cancers, and also for lung and laryngeal cancers, for both Blacks and Whites. Striking similarities in the plots for urinary bladder incidence against all three of the other cancers suggests the existence of a factor specific to the bladder. A review of black-white ratios for cancer incidences in all major body sites in both sexes and the published literature for NAT polymorphisms provided evidence that this might indeed be arylamine exposure, although other factors could also be involved.


Subject(s)
Black or African American/statistics & numerical data , Neoplasms/ethnology , Smoking/adverse effects , White People/statistics & numerical data , Arylamine N-Acetyltransferase/physiology , Cluster Analysis , Female , Humans , Incidence , Male , SEER Program , United States/epidemiology
20.
Biochem Biophys Res Commun ; 340(2): 422-7, 2006 Feb 10.
Article in English | MEDLINE | ID: mdl-16376303

ABSTRACT

Mouse ARD1 (mARD1) has been reported to negatively regulate the hypoxia-inducible factor 1alpha (HIF-1alpha) protein by acetylating a lysine residue and enhancing HIF-1alpha ubiquitination and degradation. However, it was recently reported that human ARD1 (hARD1) does not affect HIF-1alpha stability. To further explore the activities of the two orthologs, three mouse (mARD1(198), mARD1(225), mARD1(235)) and two human (hARD1(131), hARD1(235)) variants were identified and characterized. Among these, mARD1(225) was previously reported as a novel negative regulator of HIF-1alpha. Amino acid sequence analysis showed that the C-terminal region (aa 158-225) of mARD1(225) completely differs from those of mouse and human ARD1(235), although all three proteins share a well-conserved N-acetyltransferase domain (aa 45-130). The effects of ARD1 variants were evaluated with respect to HIF-1alpha stability and acetylation activity. Interestingly, mARD1(225) strongly decreased the level of HIF-1alpha and increased the extent of acetylation, whereas mARD1(235) and hARD1(235) variants had a much weaker effect on HIF-1alpha stability and acetylation. These results suggest that ARD1 variants might have different effects on HIF-1alpha stability and acetylation, which may reflect diverse biological functions that remain to be determined.


Subject(s)
Acetyltransferases/chemistry , Arylamine N-Acetyltransferase/chemistry , Isoenzymes/chemistry , Acetylation , Acetyltransferases/genetics , Acetyltransferases/physiology , Amino Acid Sequence , Animals , Arylamine N-Acetyltransferase/genetics , Arylamine N-Acetyltransferase/physiology , Cell Line, Tumor , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Isoenzymes/genetics , Isoenzymes/physiology , Mice , Molecular Sequence Data , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , NIH 3T3 Cells , RNA Stability/physiology , Sequence Homology, Amino Acid
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