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1.
J Phys Chem B ; 125(32): 9168-9185, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34351775

RESUMO

Human glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis pathway that has been extensively studied as an anticancer target. To some extent, inhibition of GAR Tfase selectively targets cancer cells over normal cells and inhibits purine formation and DNA replication. In this study, we investigated E. coli GAR Tfase, which shares high sequence similarity with the human GAR Tfase, and most functional residues are conserved. Herein, we aim to predict the pH-activity curve through a computational approach. We carried out pH-replica exchange molecular dynamics (pH-REMD) simulations to investigate pH-dependent functions such as structural changes, ligand binding, and catalytic activity. To compute the pH-activity curve, we identified the catalytic residues in specific protonation states, referred to as the catalytic competent protonation states (CCPS), which maintain the structure, keep ligands bound, and facilitate catalysis. Our computed population of CCPS with respect to pH matches well with the experimental pH-activity curve. To compute the microscopic pKa values in the catalytically active conformation, we devised a thermodynamic model that considers the coupling between protonation states of CCPS residues and conformational states. These results allow us to correctly identify the general acid and base catalysts and interpret the pH-activity curve at an atomistic level.


Assuntos
Escherichia coli , Hidroximetil e Formil Transferases , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Conformação Molecular , Fosforribosilglicinamido Formiltransferase/genética
2.
Hum Genomics ; 15(1): 25, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933170

RESUMO

BACKGROUND: Trisomy 21 (T21) is a genetic alteration characterised by the presence of an extra full or partial human chromosome 21 (Hsa21) leading to Down syndrome (DS), the most common form of intellectual disability (ID). It is broadly agreed that the presence of extra genetic material in T21 gives origin to an altered expression of genes located on Hsa21 leading to DS phenotype. The aim of this study was to analyse T21 and normal control blood cell gene expression profiles obtained by total RNA sequencing (RNA-Seq). RESULTS: The results were elaborated by the TRAM (Transcriptome Mapper) software which generated a differential transcriptome map between human T21 and normal control blood cells providing the gene expression ratios for 17,867 loci. The obtained gene expression profiles were validated through real-time reverse transcription polymerase chain reaction (RT-PCR) assay and compared with previously published data. A post-analysis through transcriptome mapping allowed the identification of the segmental (regional) variation of the expression level across the whole genome (segment-based analysis of expression). Interestingly, the most over-expressed genes encode for interferon-induced proteins, two of them (MX1 and MX2 genes) mapping on Hsa21 (21q22.3). The altered expression of genes involved in mitochondrial translation and energy production also emerged, followed by the altered expression of genes encoding for the folate cycle enzyme, GART, and the folate transporter, SLC19A1. CONCLUSIONS: The alteration of these pathways might be linked and involved in the manifestation of ID in DS.


Assuntos
Carbono-Nitrogênio Ligases/genética , Síndrome de Down/genética , Proteínas de Resistência a Myxovirus/genética , Fosforribosilglicinamido Formiltransferase/genética , Proteína Carregadora de Folato Reduzido/genética , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Cromossomos Humanos Par 21/genética , Síndrome de Down/epidemiologia , Síndrome de Down/patologia , Metabolismo Energético/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Deficiência Intelectual/epidemiologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA-Seq , Software , Transcriptoma/genética
3.
J Cell Physiol ; 235(1): 494-503, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31236962

RESUMO

Dilated cardiomyopathy (DCM) is a heart disease that injured greatly to the people wordwide. Systemic co-expression analysis for this cancer is still limited, although massive clinic experiments and gene profiling analyses had been well performed previously. Here, using the public RNA-Seq data "GSE116250" and gene annotation of Ensembl database, we built the co-expression modules for DCM by Weighted Gene Co-Expression Network Analysis, and investigated the function enrichment and protein-protein interaction (PPI) network of co-expression genes of each module by Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins database, respectively. First, 5,000 genes in the 37 samples were screened and 11 co-expression modules were conducted. The number of genes for each module ranged from 77 to 936, with a mean of 455. Second, interaction relationships of hub-genes between pairwise modules showed great differences, suggesting relatively high-scale independence of the modules. Third, functional enrichments of the co-expression modules exhibited great differences. We found that genes in module 3 were significantly enriched in the pathways of focal adhesion and ubiquitin-mediated proteolysis. This module was inferred as the key module involved in DCM. In addition, PPI analysis revealed that the genes HSP90AA1, CTNNB1, MAPK1, GART, and PPP2CA owned the largest number of adjacency genes, unveiling that they may function importantly during the occurrence of DCM. Focal adhesion and ubiquitin-mediated proteolysis play important roles in human DCM.


Assuntos
Cardiomiopatia Dilatada/genética , Adesões Focais/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Mapas de Interação de Proteínas/genética , Biomarcadores , Carbono-Nitrogênio Ligases/genética , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Miocárdio/patologia , Fosforribosilglicinamido Formiltransferase/genética , Prognóstico , Proteína Fosfatase 2/genética , beta Catenina/genética
4.
J Proteome Res ; 18(5): 2078-2087, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30964683

RESUMO

Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.


Assuntos
Amidofosforribosiltransferase/metabolismo , Mapeamento de Peptídeos/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/metabolismo , Acetilação , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Amidofosforribosiltransferase/genética , Sequência de Aminoácidos , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos/síntese química , Peptídeos/metabolismo , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , Ubiquitinação
5.
Cell Rep ; 21(5): 1331-1346, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29091770

RESUMO

Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. We find that the mTORC1 pathway is responsive to changes in purine nucleotides in a manner analogous to its sensing of amino acids. Depletion of cellular purines, but not pyrimidines, inhibits mTORC1, and restoration of intracellular adenine nucleotides via addition of exogenous purine nucleobases or nucleosides acutely reactivates mTORC1. Adenylate sensing by mTORC1 is dependent on the tuberous sclerosis complex (TSC) protein complex and its regulation of Rheb upstream of mTORC1, but independent of energy stress and AMP-activated protein kinase (AMPK). Even though mTORC1 signaling is not acutely sensitive to changes in intracellular guanylates, long-term depletion of guanylates decreases Rheb protein levels. Our findings suggest that nucleotide sensing, like amino acid sensing, enables mTORC1 to tightly coordinate nutrient availability with the synthesis of macromolecules, such as protein and nucleic acids, produced from those nutrients.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nucleotídeos de Purina/metabolismo , Células A549 , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Fluoruracila/farmacologia , Células HeLa , Humanos , Mercaptopurina/farmacologia , Metotrexato/farmacologia , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
Clin Genet ; 91(5): 748-755, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27659940

RESUMO

Congenital heart disease (CHD) is one of most prevalent birth defects in the world. However, the underlying molecular mechanism(s) have not been fully understood. Here we report that increased CHD susceptibility is associated with genetic polymorphisms for de novo nucleotide biosynthesis in northern Chinese population, which has been reported with lower plasma folate levels. Nine tagSNPs of four genes (GART, ATIC, MTHFD1 and SHMT1) in de novo nucleotide biosynthesis were sequenced in 802 sporadic CHD patients and 1093 controls from two Han Chinese populations, located in north China (Shandong) and South China (Shanghai), respectively. Six SNPs were found to be significantly associated with CHDs or septation defects only in the Shandong population dataset, but none displayed significant association with any CHDs in the Shanghai population dataset as well as in the combined dataset. We also showed that the minor A allele of rs7279549 in GART reduced transcriptional activity and displayed lower affinity for unknown transcription factor(s), demonstrating the allele is a functional risk factor for CHD in Shandong population. Our study indicates that dysregulation of de novo nucleotide biosynthesis pathway may conditionally contribute to CHD pathogenesis in northern Chinese.


Assuntos
Carbono-Nitrogênio Ligases/genética , Glicina Hidroximetiltransferase/genética , Cardiopatias Congênitas/genética , Hidroximetil e Formil Transferases/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Antígenos de Histocompatibilidade Menor/genética , Complexos Multienzimáticos/genética , Nucleotídeo Desaminases/genética , Fosforribosilglicinamido Formiltransferase/genética , Polimorfismo de Nucleotídeo Único , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Nucleotídeos/biossíntese , Nucleotídeos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
J Clin Microbiol ; 54(12): 3010-3017, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27707939

RESUMO

Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.


Assuntos
Hibridização Genômica Comparativa/métodos , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/classificação , Haemophilus influenzae/genética , Lipoproteínas/genética , Fosforribosilglicinamido Formiltransferase/genética , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA
8.
Apoptosis ; 21(12): 1386-1397, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27718035

RESUMO

Glycinamide ribonucleotide formyltransferase (GART) has been established as a pivotal enzyme in de novo purine synthesis, and mediates cellular apoptosis in many diseases. We aimed to investigate the role of GART in the pathogenesis of Crohn's disease (CD). In our study, we demonstrated for the first time that GART expression is up-regulated in patients with active CD and in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model. Moreover, the inhibition of GART induced cellular apoptosis and suppressed the migration of IECs through the activation of the MEKK3-MKK3-p38 mitogen-activated protein kinase (MAPK) pathway, following with the dys-regulation of p53 and p53 up-regulated modulator of apoptosis (PUMA). Taken together, GART plays a critical role in the protection of cellular apoptosis and migration of intestinal epithelial cells to maintain the integrity of the epithelial barrier, thus providing a new potential approach in designing a novel therapy for CD.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Colite/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Fosforribosilglicinamido Formiltransferase/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/genética , Carbono-Nitrogênio Ligases/genética , Proliferação de Células , Colite/enzimologia , Colite/genética , Colite/fisiopatologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Humanos , Intestinos/citologia , Intestinos/enzimologia , Sistema de Sinalização das MAP Quinases , Fosforribosilglicinamido Formiltransferase/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Curr Opin Virol ; 14: 30-40, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26256000

RESUMO

All known gammaherpesviruses encode at least one conserved tegument protein that contains sequence homology to the cellular purine biosynthesis enzyme: phosphoribosylformylglycineamide amidotransferase (FGARAT, or PFAS). While no enzymatic activity have been found on these viral FGARAT-homology proteins (vFGARAT), they are important for disarming host intrinsic antiviral machinery. Most vFGARAT proteins disrupt the intrinsic antiviral response-associated cellular subnuclear structure: ProMyelocytic Leukemia (PML) associated nuclear body (PML-NB). vFGARATs from different viruses target different components of PML-NB to prevent cellular repression of viral infection. In addition, vFGARATs of rhadinoviruses were recently found to oligomerize with the cellular FGARAT to deamidate RIG-I and repress inflammatory cytokine production. In this review we discuss the diverse mechanisms of antiviral response disruption by gammaherpesvirus vFGARATs and the significance of the enzyme homology domain.


Assuntos
Gammaherpesvirinae/imunologia , Gammaherpesvirinae/fisiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Proteínas Estruturais Virais/metabolismo , Humanos , Fosforribosilglicinamido Formiltransferase/genética , Proteínas Estruturais Virais/genética
10.
Tumour Biol ; 36(2): 997-1002, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25318605

RESUMO

Hepatocellular carcinoma (HCC) is the sixth common cancer and the third common cause of cancer mortality worldwide. However, the exact molecular mechanism of HCC remains uncertain. Many enzymes are involved in one-carbon metabolism (OCM), and single nucleotide polymorphisms (SNPs) in the corresponding genes may play a role in liver carcinogenesis. In this study, we enrolled 1500 HCC patients and 1500 cancer-free controls, which were frequency-matched by age, gender, and HBV infection status. Then eight SNPs from seven OCM genes (MTHFR, MTR, MTRR, FTHFD, GART, SHMT, and CBS) were evaluated. Results showed that six SNPs (MTHFR rs1801133, MTRR rs2287780, MTRR rs10380, FTHFD rs1127717, GART rs8971, and SHMT rs1979277) were significantly associated with HCC risk in Chinese population, with P values range from 2.26 × 10(-4) to 0.035). The most significant association was detected for GART rs8971. Compared with individuals with the TT genotype, the age- and sex-adjusted odds ratio (OR) for developing HCC was 1.44 (95% confidence interval (CI): 1.03-2.02) among those with the CC genotype and 1.30 (95% CI: 1.10-1.53) for those with CT genotype. Under the log-additive model, each additional copy of minor allele C was associated with a 1.28-fold increased risk of HCC (OR = 1.28, 95% CI: 1.12-1.45). These findings indicated that genetic variants in OCM genes might contribute to HCC susceptibility.


Assuntos
Carbono-Nitrogênio Ligases/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transferases de Grupo de Um Carbono/genética , Fosforribosilglicinamido Formiltransferase/genética , Adulto , Idoso , Alelos , Povo Asiático , Carcinoma Hepatocelular/patologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco
11.
Biochemistry ; 52(30): 5133-44, 2013 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-23869564

RESUMO

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.


Assuntos
Antimetabólitos Antineoplásicos/química , Carbono-Nitrogênio Ligases/química , Inibidores Enzimáticos/química , Modelos Moleculares , Fosforribosilglicinamido Formiltransferase/química , Tetra-Hidrofolatos/química , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Conformação Molecular , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Fosforribosilaminoimidazolcarboxamida Formiltransferase/química , Fosforribosilaminoimidazolcarboxamida Formiltransferase/genética , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Tetra-Hidrofolatos/metabolismo , Tetra-Hidrofolatos/farmacologia
12.
Yonsei Med J ; 54(4): 854-64, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23709418

RESUMO

PURPOSE: The main objective of this study was to evaluate the association between polymorphisms of the target genes of pemetrexed and clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with pemetrexed. MATERIALS AND METHODS: We assessed polymorphisms at 8 sites in 4 genes [thymidylate synthase (TS), dihydrofolate reductase (DHFR; 1610, 680, 317, intron 1), methylenetetrahydrofolate reductase (MTHFR; 677, 1298), glycinamide ribonucleotide formyl transferase (GARFT; 2255)] associated with pemetrexed metabolism using polymerase chain reaction, gene scanning, and restriction fragment length polymorphism analysis in 90 patients with adenocarcinoma of the lung. RESULTS: Survival was significantly longer with pemetrexed in patients with TS 3RGCC/3RGCC or 3RGGC/3RGGC compared with the other groups (PFS; 5.2 months vs. 3.7 months, p=0.03: OS; 31.8 months vs. 18.5 months, p=0.001). Patients with DHFR 680CC experienced fatigue more frequently (50% vs. 8.6%, p=0.008). Polymorphisms of MTHFR and GARFT were not significantly associated with clinical outcomes of pemetrexed. CONCLUSION: The TS genotype was associated with survival and one DHFR polymorphism was associated with fatigue in NSCLC patients treated with pemetrexed. Further large prospective studies are required to identify other biomarkers that affect patients being treated with pemetrexed for adenocarcinoma of the lung.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Adenocarcinoma/mortalidade , Adenocarcinoma de Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/toxicidade , Feminino , Glutamatos/farmacologia , Glutamatos/toxicidade , Guanina/farmacologia , Guanina/uso terapêutico , Guanina/toxicidade , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Pemetrexede , Farmacogenética , Fosforribosilglicinamido Formiltransferase/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética
13.
J Thorac Oncol ; 8(5): 644-53, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23449276

RESUMO

BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. METHODS: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. RESULTS: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. CONCLUSIONS: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Mesotelioma/enzimologia , Mesotelioma/terapia , Neoplasias Pleurais/enzimologia , Neoplasias Pleurais/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Expressão Gênica , Guanina/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Masculino , Mesotelioma/genética , Pessoa de Meia-Idade , Pemetrexede , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/genética , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Neoplasias Pleurais/genética , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Proteína Carregadora de Folato Reduzido/genética , Proteína Carregadora de Folato Reduzido/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
14.
Yonsei Medical Journal ; : 854-864, 2013.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wpr-99053

RESUMO

PURPOSE: The main objective of this study was to evaluate the association between polymorphisms of the target genes of pemetrexed and clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with pemetrexed. MATERIALS AND METHODS: We assessed polymorphisms at 8 sites in 4 genes [thymidylate synthase (TS), dihydrofolate reductase (DHFR; 1610, 680, 317, intron 1), methylenetetrahydrofolate reductase (MTHFR; 677, 1298), glycinamide ribonucleotide formyl transferase (GARFT; 2255)] associated with pemetrexed metabolism using polymerase chain reaction, gene scanning, and restriction fragment length polymorphism analysis in 90 patients with adenocarcinoma of the lung. RESULTS: Survival was significantly longer with pemetrexed in patients with TS 3RGCC/3RGCC or 3RGGC/3RGGC compared with the other groups (PFS; 5.2 months vs. 3.7 months, p=0.03: OS; 31.8 months vs. 18.5 months, p=0.001). Patients with DHFR 680CC experienced fatigue more frequently (50% vs. 8.6%, p=0.008). Polymorphisms of MTHFR and GARFT were not significantly associated with clinical outcomes of pemetrexed. CONCLUSION: The TS genotype was associated with survival and one DHFR polymorphism was associated with fatigue in NSCLC patients treated with pemetrexed. Further large prospective studies are required to identify other biomarkers that affect patients being treated with pemetrexed for adenocarcinoma of the lung.


Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adenocarcinoma/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Glutamatos/farmacologia , Guanina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Farmacogenética , Fosforribosilglicinamido Formiltransferase/genética , Polimorfismo de Nucleotídeo Único , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética
15.
Genet Test Mol Biomarkers ; 16(10): 1226-35, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22931243

RESUMO

AIM: We wished to identify markers associated with allelic nondisjunction in nuclear families with Down syndrome (DS) offspring. Since the GRIK1 and GARS-AIRS-GART genes, mapping to chromosome 21q22.1, may be informative in this regard, we genotyped four single-nucleotide polymorphisms [30952599(A/G) rs363484; 30924733(A/G) rs363506; 34901423(A/G) rs2834235; 34877070(A/G) rs7283354] present in these genes using the SNaPshot(™) assay protocol. RESULTS: We have reported 30952599(A/G)-rs363484 to be monomorphic in our sample population. Genotyping revealed 35/65 families to be informative for 34877070(A/G)-rs7283354 (GARS-AIRS-GART), whereas only 25/65 and 11/65 are informative for 34901423(A/G)-rs2834235 (GARS-AIRS-GART) and 30924733(A/G)-rs363506 (GRIK1) polymorphisms, respectively. The parent- and stage-of-origin of nondisjunction could be traced in 48/65 families using at least one polymorphic marker. A single trio provided internal validation for assignment of the parent- and stage-of-origin of nondisjunction whereby the nondisjoining alleles were independently identified as G-rs363506, G-rs2834235, and G-rs7283354, respectively. An enhanced ratio of meiosis-I to meiosis-II errors during maternal or paternal meioses accounts for allelic nondisjunction. CONCLUSIONS: The SNaPshot assay is quantitative and permits multiplexing for detection of allelic nondisjunction. Inclusion of additional informative chromosome 21-specific markers may aid rapid aneuploidy detection, screening, and prenatal counseling of parents at risk of having babies with DS.


Assuntos
Carbono-Nitrogênio Ligases/genética , Síndrome de Down/genética , Não Disjunção Genética , Fosforribosilglicinamido Formiltransferase/genética , Polimorfismo de Nucleotídeo Único , Receptores de Ácido Caínico/genética , Alelos , Cromossomos Humanos Par 21/genética , Feminino , Genótipo , Humanos , Masculino
16.
BMC Bioinformatics ; 13 Suppl 3: S3, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22536901

RESUMO

BACKGROUND: DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. RESULTS: This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing "runs" of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and ß (15%), and beta-lactamases of varying identity (26-47%). CONCLUSIONS: Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries.


Assuntos
Algoritmos , Embaralhamento de DNA , Engenharia de Proteínas/métodos , Vírus da Febre Suína Africana/enzimologia , Sequência de Aminoácidos , Animais , Bactérias/enzimologia , Códon , Escherichia coli/enzimologia , Biblioteca Gênica , Humanos , Fosforribosilglicinamido Formiltransferase/genética , Ratos , Alinhamento de Sequência , Software , beta-Lactamases/química , beta-Lactamases/genética
17.
Biol Direct ; 6: 63, 2011 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-22168471

RESUMO

BACKGROUND: The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains. RESULTS: We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function.Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified. CONCLUSIONS: The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways.


Assuntos
Archaea/genética , Genes Arqueais , Purinas/biossíntese , Archaea/química , Archaea/enzimologia , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carboxiliases/química , Carboxiliases/genética , Domínio Catalítico , Ativação Enzimática , Duplicação Gênica , Transferência Genética Horizontal , Peptídeo Sintases/química , Peptídeo Sintases/genética , Fosforribosilglicinamido Formiltransferase/química , Fosforribosilglicinamido Formiltransferase/genética , Purinas/química
18.
Cancer Lett ; 309(2): 228-35, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-21742432

RESUMO

Pemetrexed (PEM), a multitargeted antifolate with manageable toxicity, is active against non-squamous non-small cell lung cancer; however, most patients eventually acquire resistance to PEM. To elucidate the resistant mechanism, we established PEM-resistant lung adenocarcinoma cell lines. Two parental cell lines, PC-9 and A549, were treated with step-wise increasing concentrations of PEM. Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction. The four PC-9 sublines were more resistant than the PC-9 cell line to PEM (2.2-, 2.9-, 8.4-, and 14.3-fold, respectively). The four A549 sublines also showed more resistance to PEM (7.8-, 9.6-, 42.3-, and 42.4-fold, respectively) than the parent cell line. All resistant sublines showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine, 5-fluorouracil, or the active metabolite of irinotecan, SN-38. All PEM-resistant sublines expressed more TS than the parental cells, by polymerase chain reaction and Western blotting. DHFR was significantly increased in the four PEM-resistant A549 sublines. GARFT did not correlate with resistance to PEM. In summary, PEM-resistant cells remained sensitive to docetaxel, vinorelbine, 5-fluorouracil, and irinotecan. TS expression appeared to be associated with resistance to PEM.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Glutamatos/farmacologia , Guanina/análogos & derivados , Timidilato Sintase/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Proliferação de Células , Cisplatino/farmacologia , Docetaxel , Fluoruracila/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Expressão Gênica , Guanina/farmacologia , Humanos , Immunoblotting , Irinotecano , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Pemetrexede , Fosforribosilglicinamido Formiltransferase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxoides/farmacologia , Tetra-Hidrofolato Desidrogenase/biossíntese , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Vinorelbina
19.
Artigo em Inglês | MEDLINE | ID: mdl-21301108

RESUMO

Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS-PAGE and MALDI-TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Šresolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å.


Assuntos
Proteínas de Bactérias/química , Fosforribosilglicinamido Formiltransferase/química , Streptococcus mutans/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Soluções Tampão , Cristalização , Difusão , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/isolamento & purificação , Fosforribosilglicinamido Formiltransferase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Difração de Raios X , Raios X
20.
Br Poult Sci ; 51(5): 609-13, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21058063

RESUMO

1. The association of single nucleotide polymorphisms identified within the ADSL (adenylosuccinate lyase deficiency) gene and GARS-AIRS-GART (glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase) gene with the content of inosine 5'-monophosphate (IMP) was studied in a population of male Beijing-you (BJY) chickens slaughtered at 90 d of age. 2. A single nucleotide polymorphism in exon 2 of the ADSL gene had an effect on IMP content. Chickens inheriting the positive allele at ADSL, both homozygous and heterozygous genotypes, had a higher content of IMP in breast muscle than did individuals without it. 3. Similar results were obtained for the GARS-AIRS-GART gene. The marker at the 5' untranslated region (UTR) of the GARS-AIRS-GART gene was also significant for the IMP value. Chickens inheriting the genotypes with the positive allele at this locus had a much higher content of IMP than did those homozygous for the unfavourable one. 4. Interactions between ADSL and GARS-AIRS-GART were detected for such traits as body weight and muscle yields in the tested population. The two loci acted in an additive fashion. Because IMP is one of the most important flavour components in meat, markers developed at these two genes, as well as the combination genotypes, could be used as potential molecular markers for improving chicken quality.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Inosina Monofosfato/metabolismo , Polimorfismo de Nucleotídeo Único , Adenilossuccinato Liase/genética , Alelos , Animais , Peso Corporal/genética , Carbono-Nitrogênio Ligases/genética , Galinhas/metabolismo , Cromatografia Líquida de Alta Pressão , Marcadores Genéticos , Genótipo , Inosina Monofosfato/química , Masculino , Fosforribosilglicinamido Formiltransferase/genética
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