Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
1.
Pharmacogenet Genomics ; 34(7): 246-251, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38842463

RESUMO

Studies have reported overexpression of NAT1 gene for xenobiotic metabolizing arylamine N -acetyltransferase type 1 in estrogen receptor positive breast tumors, and this association has been linked to patient chemoresistance and response to tamoxifen. We probed the expression of NAT1 , using quantitative reverse transcription PCR to screen clinically characterized breast cancer tissue cDNA arrays. Primers detecting all NAT1 alternative transcripts were used, and the protocol and results are reported according to consensus guidelines. The clinical information about 166 tumor samples screened is provided, including tumor stage, estrogen and progesterone receptor status and HER2 expression. NAT1 was found to be significantly ( P  < 0.001) upregulated in hormone receptor positive vs. negative tumors. No correlation was apparent between NAT1 and tumor stage or HER2 expression. Our findings demonstrate a strong correlation between the expression of NAT1 and steroid hormone receptors in breast tumors, supporting its possible utility as a pharmacogenetic biomarker or drug target. Of the two polymorphic NAT genes, NAT1 is the one primarily expressed in breast tissue, and is subjected to regulation by two differential promoters and more than one polyadenylation signal. Hormonal factors may enhance NAT1 gene expression at the transcriptional or epigenetic level, and tamoxifen has additionally been shown to inhibit NAT1 enzymatic activity. The outcome of tamoxifen treatment is also more favorable in patients with NAT1 overexpressing tumors. The study adds to the growing body of evidence implicating NAT1 in breast cancer and its pharmacological treatment.


Assuntos
Arilamina N-Acetiltransferase , Neoplasias da Mama , Isoenzimas , Receptores de Estrogênio , Humanos , Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Isoenzimas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Tamoxifeno/uso terapêutico , Tamoxifeno/farmacologia , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
2.
Appl Environ Microbiol ; 87(19): e0081921, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34288706

RESUMO

Arylamines constitute a large group of industrial chemicals detoxified by certain bacteria through conjugation reactions catalyzed by N-acetyltransferase (NAT) enzymes. NAT homologs, mostly from pathogenic bacteria, have been the subject of individual studies that do not lend themselves to direct comparisons. By implementing a practicable pipeline, we carried out a comparative investigation of 15 NAT homologs from 10 bacteria, mainly bacilli, streptomycetes, and one alphaproteobacterium. The new homologs were characterized for their sequence, phylogeny, predicted structural features, substrate specificity, thermal stability, and interaction with components of the enzymatic reaction. Bacillus NATs demonstrated the characteristics of xenobiotic metabolizing N-acetyltransferases, with the majority of homologs generating high activities. Nonpathogenic bacilli are thus proposed as suitable mediators of arylamine bioremediation. Of the Streptomyces homologs, the NAT2 isoenzyme of S. venezuelae efficiently transformed highly toxic arylamines, while the remaining homologs were inactive or generated low activities, suggesting that xenobiotic metabolism may not be their primary role. The functional divergence of Streptomyces NATs was consistent with their observed sequence, phylogenetic, and structural variability. These and previous findings support classification of microbial NATs into three groups. The first includes xenobiotic metabolizing enzymes with dual acetyl/propionyl coenzyme A (CoA) selectivity. Homologs of the second group are more rarely encountered, acting as malonyltransferases mediating specialized ecological interactions. Homologs of the third group effectively lack acyltransferase activity, and their study may represent an interesting research area. Comparative NAT enzyme screens from a broad microbial spectrum may guide rational selection of homologs likely to share similar biological functions, allowing their combined investigation and use in biotechnological applications. IMPORTANCE Arylamines are encountered as industrial chemicals or by-products of agrochemicals that may constitute highly toxic contaminants of soils and groundwaters. Although such chemicals may be recalcitrant to biotransformation, they can be enzymatically converted into less toxic forms by some bacteria. Therefore, exploitation of the arylamine detoxification capabilities of microorganisms is investigated as an effective approach for bioremediation. Among microbial biotransformations of arylamines, enzymatic conjugation reactions have been reported, including NAT-mediated N-acetylation. Comparative investigations of NAT enzymes across a range of microorganisms can be laborious and expensive, so here we present a streamlined methodology for implementing such work. We compared 15 NAT homologs from nonpathogenic, free-living bacteria of potential biotechnological utility, mainly Terrabacteria, which are known for their rich secondary and xenobiotic metabolism. The analysis allowed insights into the evolutionary and functional divergence of bacterial NAT homologs, combined with assessment of their fundamental structural and enzymatic differences and similarities.


Assuntos
Acetiltransferases , Proteínas de Bactérias , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Xenobióticos/metabolismo
3.
World J Microbiol Biotechnol ; 35(11): 174, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673919

RESUMO

Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.


Assuntos
Actinobacteria/enzimologia , Arilamina N-Acetiltransferase/metabolismo , Actinobacteria/genética , Sequência de Aminoácidos , Aminofenóis/farmacologia , Compostos de Anilina/farmacologia , Arilamina N-Acetiltransferase/classificação , Arilamina N-Acetiltransferase/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Biotransformação , Clonagem Molecular , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Isoenzimas/genética , Cinética , Modelos Moleculares , Filogenia , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Temperatura , Xenobióticos
4.
Pharmacogenet Genomics ; 28(10): 238-244, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30222709

RESUMO

The arylamine N-acetyltransferase (NAT) nomenclature committee assigns functional phenotypes for human arylamine N-acetyltransferase 1 (NAT1) alleles in those instances in which the committee determined a consensus has been achieved in the scientific literature. In the most recent nomenclature update, the committee announced that functional phenotypes for NAT1*10 and NAT1*11 alleles were not provided owing to a lack of consensus. Phenotypic inconsistencies observed among various studies for NAT1*10 and NAT1*11 may be owing to variable allelic expression among different tissues, the limitations of the genotyping assays (which mostly relied on techniques not involving direct DNA sequencing), the differences in recombinant protein expression systems used (bacteria, yeast, and mammalian cell lines) and/or the known inherent instability of human NAT1 protein, which requires very careful handling of native and recombinant cell lysates. Three recent studies provide consistent evidence of the mechanistic basis underlying the functional phenotype of NAT1*10 and NAT1*11 as 'increased-activity' alleles. Some NAT1 variants (e.g. NAT1*14, NAT1*17, and NAT1*22) may be designated as 'decreased-activity' alleles and other NAT1 variants (e.g. NAT1*15 and NAT1*19) may be designated as 'no-activity' alleles compared with the NAT1*4 reference allele. We propose that phenotypic designations as 'rapid' and 'slow' acetylator should be discontinued for NAT1 alleles, although these designations remain very appropriate for NAT2 alleles.


Assuntos
Arilamina N-Acetiltransferase/genética , Isoenzimas/genética , Acetilação , Alelos , Regulação da Expressão Gênica/genética , Humanos , Cinética
5.
Sci Rep ; 14(1): 14905, 2024 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-38942826

RESUMO

Microbial NAT enzymes, which employ acyl-CoA to acylate aromatic amines and hydrazines, have been well-studied for their role in xenobiotic metabolism. Some homologues have also been linked to secondary metabolism, but this function of NAT enzymes is not as well-known. For this comparative study, we surveyed sequenced microbial genomes to update the list of formally annotated NAT genes, adding over 4000 new sequences (mainly bacterial, but also archaeal, fungal and protist) and portraying a broad but not universal distribution of NATs in the microbiocosmos. Localization of NAT sequences within microbial gene clusters was not a rare finding, and this association was evident across all main types of biosynthetic gene clusters (BGCs) implicated in secondary metabolism. Interrogation of the MIBiG database for experimentally characterized clusters with NAT genes further supports that secondary metabolism must be a major function for microbial NAT enzymes and should not be overlooked by researchers in the field. We also show that NAT sequences can be associated with bacterial plasmids potentially involved in horizontal gene transfer. Combined, our computational predictions and MIBiG literature findings reveal the extraordinary functional diversification of microbial NAT genes, prompting further research into their role in predicted BGCs with as yet uncharacterized function.


Assuntos
Bactérias , Família Multigênica , Metabolismo Secundário , Metabolismo Secundário/genética , Bactérias/genética , Bactérias/metabolismo , Archaea/genética , Archaea/metabolismo , Filogenia , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Fungos/genética , Genômica/métodos , Transferência Genética Horizontal
6.
BMC Evol Biol ; 13: 62, 2013 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-23497148

RESUMO

BACKGROUND: The arylamine N-acetyltransferases (NATs) are a unique family of enzymes widely distributed in nature that play a crucial role in the detoxification of aromatic amine xenobiotics. Considering the temporal changes in the levels and toxicity of environmentally available chemicals, the metabolic function of NATs is likely to be under adaptive evolution to broaden or change substrate specificity over time, making NATs a promising subject for evolutionary analyses. In this study, we trace the molecular evolutionary history of the NAT gene family during the last ~450 million years of vertebrate evolution and define the likely role of gene duplication, gene conversion and positive selection in the evolutionary dynamics of this family. RESULTS: A phylogenetic analysis of 77 NAT sequences from 38 vertebrate species retrieved from public genomic databases shows that NATs are phylogenetically unstable genes, characterized by frequent gene duplications and losses even among closely related species, and that concerted evolution only played a minor role in the patterns of sequence divergence. Local signals of positive selection are detected in several lineages, probably reflecting response to changes in xenobiotic exposure. We then put a special emphasis on the study of the last ~85 million years of primate NAT evolution by determining the NAT homologous sequences in 13 additional primate species. Our phylogenetic analysis supports the view that the three human NAT genes emerged from a first duplication event in the common ancestor of Simiiformes, yielding NAT1 and an ancestral NAT gene which in turn, duplicated in the common ancestor of Catarrhini, giving rise to NAT2 and the NATP pseudogene. Our analysis suggests a main role of purifying selection in NAT1 protein evolution, whereas NAT2 was predicted to mostly evolve under positive selection to change its amino acid sequence over time. These findings are consistent with a differential role of the two human isoenzymes and support the involvement of NAT1 in endogenous metabolic pathways. CONCLUSIONS: This study provides unequivocal evidence that the NAT gene family has evolved under a dynamic process of birth-and-death evolution in vertebrates, consistent with previous observations made in fungi.


Assuntos
Arilamina N-Acetiltransferase/genética , Evolução Molecular , Família Multigênica , Seleção Genética , Animais , Ordem dos Genes , Humanos , Isoenzimas , Funções Verossimilhança , Filogenia , Recombinação Genética , Alinhamento de Sequência , Vertebrados/genética
7.
FEBS J ; 290(9): 2412-2436, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36178468

RESUMO

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.


Assuntos
Arilamina N-Acetiltransferase , Fusarium , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/genética , Fusarium/genética , Isoenzimas/genética , Filogenia , Acetilcoenzima A , Acetiltransferases
8.
Front Pharmacol ; 14: 1278720, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38035025

RESUMO

Introduction: Several polymorphisms altering the NAT2 activity have already been identified. The geographical distribution of NAT2 variants has been extensively studied and has been demonstrated to vary significantly among different ethnic population. Here, we describe the genetic variability of human N-acetyltransferase 2 (NAT2) gene and the predominant genotype-deduced acetylation profiles of Brazilians. Methods: A total of 964 individuals, from five geographical different regions, were genotyped for NAT2 by sequencing the entire coding exon. Results: Twenty-three previously described NAT2 single nucleotide polymorphisms (SNPs) were identified, including the seven most common ones globally (c.191G>A, c.282C>T, c.341T>C, c.481C>T, c.590G>A, c.803A>G and c.857G>A). The main allelic groups were NAT2*5 (36%) and NAT2*6 (18.2%), followed to the reference allele NAT2*4 (20.4%). Combined into genotypes, the most prevalent allelic groups were NAT2*5/*5 (14.6%), NAT2*5/*6 (11.9%) and NAT2*6/*6 (6.2%). The genotype deduced NAT2 slow acetylation phenotype was predominant but showed significant variability between geographical regions. The prevalence of slow acetylation phenotype was higher in the Northeast, North and Midwest (51.3%, 45.5% and 41.5%, respectively) of the country. In the Southeast, the intermediate acetylation phenotype was the most prevalent (40.3%) and, in the South, the prevalence of rapid acetylation phenotype was significantly higher (36.7%), when compared to other Brazilian states (p < 0.0001). Comparison of the predicted acetylation profile among regions showed homogeneity among the North and Northeast but was significantly different when compared to the Southeast (p = 0.0396). The Southern region was significantly different from all other regions (p < 0.0001). Discussion: This study contributes not only to current knowledge of the NAT2 population genetic diversity in different geographical regions of Brazil, but also to the reconstruction of a more accurate phenotypic picture of NAT2 acetylator profiles in those regions.

9.
PLoS One ; 17(7): e0271125, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35834592

RESUMO

Bacteria employ secondary metabolism to combat competitors, and xenobiotic metabolism to survive their chemical environment. This project has aimed to introduce a bacterial collection enabling comprehensive comparative investigations of those functions. The collection comprises 120 strains (Proteobacteria, Actinobacteria and Firmicutes), and was compiled on the basis of the broad taxonomic range of isolates and their postulated biosynthetic and/or xenobiotic detoxification capabilities. The utility of the collection was demonstrated in two ways: first, by performing 5144 co-cultures, recording inhibition between isolates and employing bioinformatics to predict biosynthetic gene clusters in sequenced genomes of species; second, by screening for xenobiotic sensitivity of isolates against 2-benzoxazolinone and 2-aminophenol. The co-culture medium of Bacillus siamensis D9 and Lysinibacillus sphaericus DSM 28T was further analysed for possible antimicrobial compounds, using liquid chromatography-mass spectrometry (LC-MS), and guided by computational predictions and the literature. Finally, LC-MS analysis demonstrated N-acetylation of 3,4-dichloroaniline (a toxic pesticide residue of concern) by the actinobacterium Tsukamurella paurometabola DSM 20162T which is highly tolerant of the xenobiotic. Microbial collections enable "pipeline" comparative screening of strains: on the one hand, bacterial co-culture is a promising approach for antibiotic discovery; on the other hand, bioremediation is effective in combating pollution, but requires knowledge of microbial xenobiotic metabolism. The presented outcomes are anticipated to pave the way for studies that may identify bacterial strains and/or metabolites of merit in biotechnological applications.


Assuntos
Bactérias , Xenobióticos , Firmicutes , Proteobactérias , Metabolismo Secundário
10.
Genome Biol Evol ; 14(12)2022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36445690

RESUMO

Variation in genes involved in the absorption, distribution, metabolism, and excretion of drugs (ADME) can influence individual response to a therapeutic treatment. The study of ADME genetic diversity in human populations has led to evolutionary hypotheses of adaptation to distinct chemical environments. Population differentiation in measured drug metabolism phenotypes is, however, scarcely documented, often indirectly estimated via genotype-predicted phenotypes. We administered seven probe compounds devised to target six cytochrome P450 enzymes and the P-glycoprotein (P-gp) activity to assess phenotypic variation in four populations along a latitudinal transect spanning over Africa, the Middle East, and Europe (349 healthy Ethiopian, Omani, Greek, and Czech volunteers). We demonstrate significant population differentiation for all phenotypes except the one measuring CYP2D6 activity. Genome-wide association studies (GWAS) evidenced that the variability of phenotypes measuring CYP2B6, CYP2C9, CYP2C19, and CYP2D6 activity was associated with genetic variants linked to the corresponding encoding genes, and additional genes for the latter three. Instead, GWAS did not indicate any association between genetic diversity and the phenotypes measuring CYP1A2, CYP3A4, and P-gp activity. Genome scans of selection highlighted multiple candidate regions, a few of which included ADME genes, but none overlapped with the GWAS candidates. Our results suggest that different mechanisms have been shaping the evolution of these phenotypes, including phenotypic plasticity, and possibly some form of balancing selection. We discuss how these contrasting results highlight the diverse evolutionary trajectories of ADME genes and proteins, consistent with the wide spectrum of both endogenous and exogenous molecules that are their substrates.


Assuntos
Citocromo P-450 CYP2D6 , Estudo de Associação Genômica Ampla , Humanos , Citocromo P-450 CYP2D6/genética , Xenobióticos , Fenótipo , Genômica
12.
Biochem Pharmacol ; 188: 114545, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33831395

RESUMO

Human NAT2 is a polymorphic pharmacogene encoding for N-acetyltransferase 2, a hepatic enzyme active towards arylamine and arylhydrazine drugs, including the anti-tubercular antibiotic isoniazid. The isoenzyme also modulates susceptibility to chemical carcinogenesis, particularly of the bladder. Human NAT2 represents an ideal model for anthropological investigations into the demographic adaptation of worldwide populations to their xenobiotic environment. Its sequence appears to be subject to positive selection pressures that are population-specific and may be attributed to gene-environment interactions directly associated with exogenous chemical challenges. However, recent evidence suggests that the same evolutionary pattern may not be observed in other primates. Here, we report NAT2 polymorphism in 25 rhesus macaques (Macaca mulatta) and compare the frequencies and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified, including one nonsense mutation. The missense SNVs were demonstrated to affect enzymatic function in a substrate-dependent manner, albeit more moderately than certain NAT1 SNVs recently characterised in the same cohort. Haplotypic and functional variability of NAT2 was comparable to that previously observed for NAT1 in the same population sample, suggesting that the two paralogues may have evolved under similar selective pressures in the rhesus macaque. This is different to the population variability distribution pattern reported for humans and chimpanzees. Recorded SNVs were also different from those found in other primates. The study contributes to further understanding of NAT2 functional polymorphism in the rhesus macaque, a non-human primate model used in biomedicine and pharmacology, indicating variability in xenobiotic acetylation that could affect drug metabolism.


Assuntos
Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Variação Genética/fisiologia , Polimorfismo Genético/fisiologia , Sequência de Aminoácidos , Animais , Antituberculosos/farmacologia , Arilamina N-Acetiltransferase/química , Variação Genética/efeitos dos fármacos , Humanos , Isoniazida/farmacologia , Macaca mulatta , Polimorfismo Genético/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
Clin Cancer Res ; 14(8): 2465-75, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18413839

RESUMO

PURPOSE: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. EXPERIMENTAL DESIGN: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. RESULTS: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. CONCLUSIONS: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.


Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica , Mutação , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Humanos , Lapatinib , Fosforilação , Conformação Proteica , Receptor ErbB-2/química
15.
Sci Rep ; 9(1): 10937, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358821

RESUMO

Human NAT1 gene for N-acetyltransferase 1 modulates xenobiotic metabolism of arylamine drugs and mutagens. Beyond pharmacogenetics, NAT1 is also relevant to breast cancer. The population history of human NAT1 suggests evolution through purifying selection, but it is unclear whether this pattern is evident in other primate lineages where population studies are scarce. We report NAT1 polymorphism in 25 rhesus macaques (Macaca mulatta) and describe the haplotypic and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified and experimentally demonstrated to compromise enzyme function, mainly through destabilization of NAT1 protein and consequent activity loss. One non-synonymous SNV (c.560G > A, p.Arg187Gln) has also been characterized for human NAT1 with similar effects. Population haplotypic and functional variability of rhesus NAT1 was considerably higher than previously reported for its human orthologue, suggesting different environmental pressures in the two lineages. Known functional elements downstream of human NAT1 were also differentiated in rhesus macaque and other primates. Xenobiotic metabolizing enzymes play roles beyond mere protection from exogenous chemicals. Therefore, any link to disease, particularly carcinogenesis, may be via modulation of xenobiotic mutagenicity or more subtle interference with cell physiology. Comparative analyses add the evolutionary dimension to such investigations, assessing functional conservation/diversification among primates.


Assuntos
Arilamina N-Acetiltransferase/genética , Isoenzimas/genética , Polimorfismo de Nucleotídeo Único , Animais , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Estabilidade Enzimática , Evolução Molecular , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Macaca mulatta , Mutação , Xenobióticos/metabolismo
16.
Curr Drug Metab ; 9(7): 628-60, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18781915

RESUMO

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes found in prokaryotes and eukaryotes. NATs have been characterized in bacteria (Bacilli, Mycobacteria, Salmonella etc.), laboratory animals (chicken, rabbit, rodents etc.) and humans, where the NAT loci occupy 230 kilobases on chromosome 8p22. Our previous comprehensive search for NAT genes involved 416 genomes (340 prokaryotic, 76 eukaryotic) and identified NAT homologues in several taxa, while also reporting on taxa that appeared to lack NAT genes [Boukouvala, S. and Fakis, G. (2005) Drug Metab. Rev. 37(3), 511-564]. Here, we present an update of this genomic search, covering 2138 genomes (1674 prokaryotic, 464 eukaryotic), of which 1167 (986 prokaryotic, 181 eukaryotic) were accessible using the advanced search algorithm tBLASTn. We have reconstructed the full-length open reading frames for putative proteins with sequence homology and features characteristic of NAT from 274 bacterial genomes (31 actinobacteria, 6 bacteroidetes/chlorobi, 2 cyanobacteria, 65 firmicutes and 170 proteobacteria) and 27 animals (1 sea-urchin, 5 fishes, 1 lizard, 1 bird and 19 mammals). Partial NAT sequences were recovered from several other organisms, including fungi, where NAT genes were found in 30 ascomycetes and 2 basidiomycetes. No NATs were found in arhaea, plants and lower invertebrates (insects and worms), while it is also uncertain whether NAT genes exist in protista. We present comparative genomic and phylogenetic analyses of the identified NAT homologues and announce a new database that will maintain information on non-human NATs and will provide recommendations for a standardized nomenclature, along the lines of the NAT Gene Nomenclature Committee.


Assuntos
Archaea/enzimologia , Arilamina N-Acetiltransferase/genética , Bactérias/enzimologia , Fungos/enzimologia , Genoma , Animais , Archaea/genética , Arilamina N-Acetiltransferase/classificação , Arilamina N-Acetiltransferase/metabolismo , Bactérias/genética , Bases de Dados como Assunto , Fungos/genética , Humanos , Filogenia , Terminologia como Assunto
17.
Drug Metab Rev ; 40(3): 479-510, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18642144

RESUMO

Arylamine N-acetyltransferases (NATs) are cytosolic conjugating enzymes which transfer an acetyl group from acetylCoenzyme A to a xenobiotic acceptor substrate. The enzyme has an active site cysteine as part of a catalytic triad with histidine and aspartate. NATs have had an important role in pharmacogenetics. Polymorphism in acetylation (and inactivation) of the anti-tubercular agent isoniazid resides in human NAT2, one of two polymorphic human NATs. In humans there is also a third pseudogene and in rodents there are three isozymes. Comparison of human and rodent NAT enzymes and their genes is aiding our understanding of the roles of the individual isoenzymes. This may have clinical importance since human NAT1 is overexpressed in a sub-population of breast cancers and control of expression of the NAT genes is ripe for investigation. The mammalian NAT enzymes are involved in metabolism of drugs and carcinogens but there is growing evidence, including from transgenic mice, that human NAT1 has an endogenous role in folate degradation. Structural studies and intracellular tracking of polymorphic NAT variants, is contributing to appreciation of how individual mutations result in loss of NAT activity. Genome analyses have identified NAT homologues in bacteria including Mycobacterium tuberculosis, in which the NAT enzyme metabolises inactivation of isoniazid. More intriguingly, deletion of the nat gene in mycobacteria, leads to deficits in cell wall synthesis. Structural comparisons of NATs from prokaryotes and eukaryotes, particularly in relation to CoA binding, provide a platform for understanding how the unique NAT protein fold may lend itself to a wide range of functions.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Antituberculosos/metabolismo , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Citosol/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Isoniazida/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Farmacogenética , Polimorfismo Genético , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Sci Rep ; 8(1): 9759, 2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29950659

RESUMO

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates.


Assuntos
Acetiltransferases/metabolismo , Isoenzimas/metabolismo , Animais , Humanos , Macaca , Primatas , Especificidade por Substrato
19.
Biochem J ; 375(Pt 3): 593-602, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12904181

RESUMO

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.


Assuntos
Arilamina N-Acetiltransferase/genética , Transcrição Gênica/genética , Animais , Arilamina N-Acetiltransferase/metabolismo , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Feminino , Regulação Enzimológica da Expressão Gênica , Genes/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
20.
Basic Clin Pharmacol Toxicol ; 96(5): 343-51, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15853926

RESUMO

Arylamine N-acetyltransferases are polymorphic drug-metabolising enzymes. The human isoforms, NAT1 and NAT2, are encoded by two genes with intronless coding regions. Human NAT1 protein is found in many tissues, unlike NAT2 which is present predominantly in the intestine and liver. We describe the exon-intron structure of the human NAT genes by analysing data from genomic databases. Comparison of expressed sequence tags, matching NAT gene sequences, with the sequence of human chromosome 8 implied the presence of 8 non-coding exons located 51.5, 51.4, 12.3, 11.9, 10.8, 9.6, 5.2 and 2.6 kb upstream of the single coding exon of the NAT1 gene. A number of expressed sequence tags also indicated transcription initiation from the upstream region adjacent to the NAT1 coding exon, consistent with earlier studies. The NAT2 gene consists of one previously described non-coding and one coding exon, located 8.6 kb apart. These findings were also confirmed by RT-PCR, using cDNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Alternatively spliced NAT1 transcripts were found in all tissues. Transcription of the NAT2 gene was also detected in these tissues and was demonstrated to start either from the non-coding exon or from immediately upstream of the coding exon. Comparison of the RT-PCR products provided an initial estimate of the relative amounts of the different NAT transcripts expressed in each tissue. Finally, both expressed sequence tag analysis and RT-PCR demonstrated the presence of two differentially utilised polyadenylation signals for NAT1 and NAT2, located about 0.2 and 0.3 kb downstream of the coding region of each gene.


Assuntos
Processamento Alternativo , Arilamina N-Acetiltransferase/genética , Éxons/genética , Íntrons/genética , Transcrição Gênica/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 8/genética , Etiquetas de Sequências Expressas , Genes/genética , Biblioteca Genômica , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , Poliadenilação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA