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1.
Nat Med ; 25(5): 850-860, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068703

RESUMO

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.


Assuntos
Neoplasias/metabolismo , Animais , Asparaginase/uso terapêutico , Asparagina/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/antagonistas & inibidores , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cinurenina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Metaboloma , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/terapia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/terapia
2.
Gene ; 704: 97-102, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30978478

RESUMO

In the current study, we report three cases of Asparagine Synthetase (ASNS) Deficiency from two consanguineous families. Family 1 had two early neonatal deaths due to a novel mutation in the ASNS gene c.788C > T (p.S263F) and both the children presented with microcephaly and one of them had severe intracranial haemorrhage. The proband from the second family was homozygous for c.146G > A (p.R49Q) and manifested myoclonic seizures, developmental delay, coarse hair and diffuse cortical atrophy. Molecular docking studies of both the mutations revealed alteration in the ligand binding site. Till date, 26 mutations were reported in ASNS gene in 29 affected children indicating high degree of genetic heterogeneity and high mortality. Although asparagine depletion is not of diagnostic utility, multiple linear regression model suggested that asparagine levels vary to the extent of 20.6% based on glutamine and aspartate levels and ASNS deficiency results in depletion of asparagine synthesis. ASNS deficiency should be suspected in any neonate with microcephaly and epileptic encephalopathy.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Deficiências do Desenvolvimento/genética , Microcefalia/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Grupo com Ancestrais do Continente Asiático , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/deficiência , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/patologia , Família , Feminino , Humanos , Índia , Lactente , Recém-Nascido , Hemorragias Intracranianas/congênito , Hemorragias Intracranianas/genética , Masculino , Microcefalia/patologia , Técnicas de Diagnóstico Molecular , Morte Perinatal , Convulsões/complicações , Convulsões/genética
3.
Nutrients ; 11(1)2019 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-30609725

RESUMO

The aim of this study was to determine if increased mortality associated with low levels of serum 25-hydroxyvitamin D (25(OH)D) reflects a causal relationship by using a Mendelian randomisation (MR) approach with genetic variants in the vitamin D synthesis pathway. Individual participant data from three European cohorts were harmonized with standardization of 25(OH)D according to the Vitamin D Standardization Program. Most relevant single nucleotide polymorphisms of the genes CYP2R1 (rs12794714, rs10741657) and DHCR7/NADSYN1 (rs12785878, rs11234027), were combined in two allelic scores. Cox proportional hazards regression models were used with the ratio estimator and the delta method for calculating the hazards ratio (HR) and standard error of genetically determined 25(OH)D effect on all-cause mortality. We included 10,501 participants (50.1% females, 67.1±10.1 years) of whom 4003 died during a median follow-up of 10.4 years. The observed adjusted HR for all-cause mortality per decrease in 25(OH)D by 20 nmol/L was 1.20 (95% CI: 1.15⁻1.25). The HR per 20 nmol/L decrease in genetically determined 25(OH)D was 1.32 (95% CI: 0.80⁻2.24) and 1.35 (95% CI of 0.81 to 2.37) based on the two scores. In conclusion, the results of this MR study in a combined sample from three European cohort studies provide further support for a causal relationship between vitamin D deficiency and increased all-cause mortality. However, as the current study, even with ~10,000 participants, was underpowered for the study of the effect of the allele score on mortality, larger studies on genetics and mortality are needed to improve the precision.


Assuntos
Predisposição Genética para Doença , Análise da Randomização Mendeliana , Vitamina D/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Estudos de Coortes , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Europa (Continente) , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Polimorfismo de Nucleotídeo Único , Vitamina D/sangue
4.
J Neurosci Res ; 96(2): 297-304, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28834557

RESUMO

BACKGROUND: MS is a neurodegenerative autoimmune disease resulting from a complex interaction of genetic and environmental factors. Among these, vitamin D and genetic variants associated with vitamin D-metabolism gain great attention. The aim of our study was to assess five SNPs in NADSYN1 and CYP2R1 genes in relation to serum 25-OH-vitamin D3 levels in MS patients and controls. METHODS: 25-OH-vitamin D3 levels and genotyping of CYP2R1- and NADSYN1-SNPs were investigated both in MS patients and in healthy controls. RESULTS: The analysis revealed lower 25-OH-vitamin D3 concentrations in MS patients than in controls and an association of rs10766197 CYP2R1 SNP with MS risk. After stratifying MS patients according to gender, we found that the minor allele A of rs10766197 had a higher frequency in men in comparison to women affected by MS. Additionally, the presence of allele A in men was associated with disease progression, assessed by EDSS and MSSS scores. CONCLUSION: The findings of our study open new perspectives for a role of CYP2R1 in both risk and progression of MS, with sex-related differences.


Assuntos
Colestanotriol 26-Mono-Oxigenase/genética , Família 2 do Citocromo P450/genética , Predisposição Genética para Doença/genética , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Estudos de Casos e Controles , Avaliação da Deficiência , Progressão da Doença , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Estudos Retrospectivos , Índice de Gravidade de Doença , Fatores Sexuais , Vitamina D/sangue
5.
Viruses ; 9(10)2017 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-29065450

RESUMO

Gammaherpesviruses like Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS), an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML) protein-associated nuclear bodies (PML-NBs) by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.


Assuntos
Herpesvirus Humano 8/química , Interações Hospedeiro-Patógeno , Proteína da Leucemia Promielocítica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Replicação do DNA/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/enzimologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/genética , Ubiquitinação , Replicação Viral
6.
J Dairy Sci ; 100(10): 8176-8187, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28803020

RESUMO

A candidate mutation in the sex hormone binding globulin gene was proposed in 2013 to be responsible for the MH1 recessive embryonic lethal locus segregating in the Montbéliarde breed. In this follow-up study, we excluded this candidate variant because healthy homozygous carriers were observed in large-scale genotyping data generated in the framework of the genomic selection program. We fine mapped the MH1 locus in a 702-kb interval and analyzed genome sequence data from the 1,000 bull genomes project and 54 Montbéliarde bulls (including 14 carriers and 40 noncarriers). We report the identification of a strong candidate mutation in the gene encoding phosphoribosylformylglycinamidine synthase (PFAS), a protein involved in de novo purine synthesis. This mutation, located in a class I glutamine amidotransferase-like domain, results in the substitution of an arginine residue that is entirely conserved among eukaryotes by a cysteine (p.R1205C). No homozygote for the cysteine-encoding allele was observed in a large population of more than 25,000 individuals despite a 6.7% allelic frequency and 122 expected homozygotes under neutrality assumption. Genotyping of 18 embryos collected from heterozygous parents as well as analysis on nonreturn rates suggested that most homozygous carriers died between 7 and 35 d postinsemination. The identification of this strong candidate mutation will enable the accurate testing of the reproducers and the efficient selection against this lethal recessive embryonic defect in the Montbéliarde breed.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Genótipo , Haplótipos , Mutação de Sentido Incorreto , Animais , Cruzamento , Bovinos , Seguimentos , Masculino , Especificidade da Espécie
7.
Hum Mol Genet ; 26(5): 1003-1017, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062664

RESUMO

Studies attempting to functionally interpret complex-disease susceptibility loci by GWAS and eQTL integration have predominantly employed microarrays to quantify gene-expression. RNA-Seq has the potential to discover a more comprehensive set of eQTLs and illuminate the underlying molecular consequence. We examine the functional outcome of 39 variants associated with Systemic Lupus Erythematosus (SLE) through the integration of GWAS and eQTL data from the TwinsUK microarray and RNA-Seq cohort in lymphoblastoid cell lines. We use conditional analysis and a Bayesian colocalisation method to provide evidence of a shared causal-variant, then compare the ability of each quantification type to detect disease relevant eQTLs and eGenes. We discovered the greatest frequency of candidate-causal eQTLs using exon-level RNA-Seq, and identified novel SLE susceptibility genes (e.g. NADSYN1 and TCF7) that were concealed using microarrays, including four non-coding RNAs. Many of these eQTLs were found to influence the expression of several genes, supporting the notion that risk haplotypes may harbour multiple functional effects. Novel SLE associated splicing events were identified in the T-reg restricted transcription factor, IKZF2, and other candidate genes (e.g. WDFY4) through asQTL mapping using the Geuvadis cohort. We have significantly increased our understanding of the genetic control of gene-expression in SLE by maximising the leverage of RNA-Seq and performing integrative GWAS-eQTL analysis against gene, exon, and splice-junction quantifications. We conclude that to better understand the true functional consequence of regulatory variants, quantification by RNA-Seq should be performed at the exon-level as a minimum, and run in parallel with gene and splice-junction level quantification.


Assuntos
Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Locos de Características Quantitativas/genética , RNA não Traduzido/genética , Processamento Alternativo/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/biossíntese , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Mapeamento Cromossômico , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Lúpus Eritematoso Sistêmico/patologia , Masculino , Polimorfismo de Nucleotídeo Único , Fator 1 de Transcrição de Linfócitos T/biossíntese , Fator 1 de Transcrição de Linfócitos T/genética
8.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 8): 627-35, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27487927

RESUMO

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Methanocaldococcus/química , Sulfolobus/química , Thermus thermophilus/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Methanocaldococcus/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologia , Thermus thermophilus/enzimologia
9.
Med Sci Monit ; 21: 1960-8, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26149120

RESUMO

BACKGROUND: Our study is aimed to 1) clarify the vitamin D status in Uygur and Kazak ethnic populations and 2) elucidate the relationship between 14 SNPs (in 5 vitamin D-related genes) and vitamin D deficiency in these 2 ethnic populations. MATERIAL AND METHODS: A multistage-cluster sampling survey was carried out for residents with Uygur or Kazak ethnicity in Xinjiang, China. Anthropometric measurements were taken and the concentrations of 25OHD were measured. Fourteen common variants in VDR, GC, CYP2R1, CYP27B1, and DHCR7/NADSYN1 were genotyped by using multiple SNaPshot assay. Logistic regression analysis was performed to identify the possible risk factors for vitamin D deficiency, after adjusting for several environmental and biological factors. The pattern of SNP associations was distinct between Uygurs and Kazaks. RESULTS: Anthropometric measurements and the concentrations of 25OHD were obtained from 1873 participants (945 Uygur ethnic and 928 Kazak ethnic). The genotypes of 14 SNPs were measured for 300 Uygurs and 300 Kazaks. The median 25OHD concentration was as low as 10.4 ng/ml in Uygurs and 16.2 ng/ml in Kazaks. In Uygurs, the prevalence of vitamin D deficiency, in-sufficiency, and sufficiency was 91.2%, 5.8%, and 3.0%, respectively. CYP2R1-rs10766197 was significantly associated with the presence of vitamin D deficiency in the Uygur ethnic population (P=0.019, OR=6.533, 95%C.I.: 361-31.357), while DHCR7/NADSYN1-rs12785878 was significantly associated with the presence of vitamin D deficiency in the Kazak ethnic population (P=0.011, OR=2.442, 95%C.I.: 1.224-4.873). Of 10 SNPs in VDR and GC genes, none was associated with vitamin D status in these 2 ethnic populations. CONCLUSIONS: Vitamin D insufficiency is highly prevalent in Uygurs and Kazaks living in Xinjiang, China. Polymorphisms in CYP2R1-rs10766197 and DHCR7/NADSYN1-rs12785878 are associated with vitamin D deficiency in Uygur and Kazak ethnic populations.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Colestanotriol 26-Mono-Oxigenase/genética , Grupos Étnicos/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Polimorfismo de Nucleotídeo Único , Deficiência de Vitamina D/genética , Adulto , China , Família 2 do Citocromo P450 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
Mol Cell ; 58(1): 134-46, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25752576

RESUMO

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here, we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologs of phosphoribosylformylglycinamidine synthetase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homolog thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/imunologia , RNA Helicases DEAD-box/imunologia , Gammaherpesvirinae/imunologia , Evasão da Resposta Imune/genética , RNA Viral/imunologia , Proteínas Virais/imunologia , Amidas/metabolismo , Animais , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Linhagem Celular , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Proteína DEAD-box 58 , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Ativação Enzimática , Fibroblastos/enzimologia , Fibroblastos/imunologia , Fibroblastos/virologia , Gammaherpesvirinae/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Camundongos , Mimetismo Molecular , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Transdução de Sinais , Proteínas Virais/genética
11.
Biochemistry ; 52(31): 5225-35, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23841499

RESUMO

Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Domínio Catalítico , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanosina Monofosfato/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ribonucleotídeos/metabolismo , Especificidade por Substrato
12.
J Nutrigenet Nutrigenomics ; 6(6): 327-35, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24642724

RESUMO

BACKGROUND/AIMS: Recent genome-wide association studies have identified the rs1790349 and rs12785878 single-nucleotide polymorphisms (SNPs), present in the NADSYN1/DHCR7 locus, as an influential player on circulating 25-hydroxyvitamin D [25(OH)D] levels, which itself has been linked to various diseases including cardiovascular disease (CVD). This study investigated the association of these SNPs with CVD and 25(OH)D levels. METHODS: Sixty- three male patients with verified coronary artery disease (CAD) were recruited, as well as 31 age- and sex-matched controls. Genotyping was performed by sequencing, whereas plasma 25(OH)D levels were assessed by HPLC-UV. RESULTS: Statistical insignificance was observed in comparing the genotype distribution of patients and controls for both the rs12785878 (NADSYN1) polymorphism (p = 0.097) and the rs1790349 (DHCR7; p = 0.9). Comparison of allelic distributions of rs1790349 and rs12785878 yielded insignificant results (p = 0.7, OR: 0.58-2.6 and p = 0.14, OR: 0.88-2.85, respectively). Taking together patients and controls, both SNPs were found to influence total 25(OH)D levels (p = 0.001 and p < 0.0001) as well as 25(OH)D3 levels only in controls. CONCLUSION: This study further supports the evidence of the ability of the investigated SNPs to predict circulating 25(OH)D levels, nonetheless opposing their use as genetic markers for CAD.


Assuntos
Amida Sintases/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Doença da Artéria Coronariana/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Polimorfismo Genético , Vitamina D/análogos & derivados , Adulto , Alelos , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Incidência , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Vitamina D/sangue
13.
Mol Cell Biol ; 32(13): 2396-406, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22508989

RESUMO

The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteínas Fúngicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidil Transferases/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sequência de Bases , Sítios de Ligação , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Sistema Livre de Células , Códon de Terminação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Neurospora crassa/genética , Neurospora crassa/metabolismo , Fases de Leitura Aberta , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptidil Transferases/antagonistas & inibidores , Peptidil Transferases/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
14.
J Mol Biol ; 416(4): 518-33, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22244852

RESUMO

The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.


Assuntos
Arginina/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Proteínas Fúngicas/química , Fragmentos de Peptídeos/química , Proteínas Ribossômicas/química , Ribossomos/química , Sequência de Aminoácidos , Sequência de Bases , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Dados de Sequência Molecular , Mutação , Neurospora/química , Fases de Leitura Aberta , Fragmentos de Peptídeos/genética , Conformação Proteica
15.
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
16.
Genetics ; 188(2): 359-67, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21441212

RESUMO

The biosynthetic pathways and multiple functions of purine nucleotides are well known. However, the pathways that respond to alterations in purine nucleotide synthesis in vivo in an animal model organism have not been identified. We examined the effects of inhibiting purine de novo synthesis in vivo and in cultured cells of Drosophila melanogaster. The purine de novo synthesis gene ade2 encodes phosphoribosylformylglycinamidine synthase (EC 6.3.5.3). An ade2 deletion, generated by P-element transposon excision, causes lethality in early pupal development, with darkening, or necrosis, of leg and wing imaginal disc tissue upon disc eversion. Together with analysis of a previously isolated weaker allele, ade2(4), and an allele of the Prat gene, which encodes an enzyme for the first step in the pathway, we determined that the lethal arrest and imaginal disc phenotypes involve apoptosis. A transgene expressing the baculovirus caspase inhibitor p35, which suppresses apoptosis caused by other stresses such as DNA damage, suppresses both the imaginal disc tissue darkening and the pupal lethality of all three purine de novo synthesis mutants. Furthermore, we showed the presence of apoptosis at the cellular level in both ade2 and Prat mutants by detecting TUNEL-positive nuclei in wing imaginal discs. Purine de novo synthesis inhibition was also examined in tissue culture by ade2 RNA interference followed by analysis of genome-wide changes in transcript levels. Among the upregulated genes was HtrA2, which encodes an apoptosis effector and is thus a candidate for initiating apoptosis in response to purine depletion.


Assuntos
Apoptose , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Nucleotídeos de Purina/biossíntese , Amidofosforribosiltransferase/genética , Amidofosforribosiltransferase/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Vias Biossintéticas , Western Blotting , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Linhagem Celular , Cruzamentos Genéticos , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Feminino , Perfilação da Expressão Gênica , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Marcação In Situ das Extremidades Cortadas , Masculino , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Pupa/genética , Pupa/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
17.
Plant J ; 65(3): 335-45, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21265888

RESUMO

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arginina/biossíntese , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Mutação , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Citrulina/genética , Citrulina/metabolismo , Citrulina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Metanossulfonato de Etila/farmacologia , Células do Mesofilo/metabolismo , Metabolômica , Dados de Sequência Molecular , Morfogênese/genética , Ornitina/genética , Ornitina/metabolismo , Ornitina/farmacologia , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Alinhamento de Sequência
18.
J Biol Chem ; 285(52): 40933-42, 2010 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20884617

RESUMO

The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9-20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Substituição de Aminoácidos , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Proteínas Fúngicas/genética , Mutação de Sentido Incorreto , Neurospora crassa/genética , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , RNA Fúngico/genética , RNA Fúngico/metabolismo , Ribossomos/genética
19.
Mol Cell ; 40(1): 138-46, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20932481

RESUMO

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Citomegalovirus/metabolismo , Fragmentos de Peptídeos/química , Biossíntese de Proteínas , Ribossomos/ultraestrutura , Proteínas do Envelope Viral/química , Leveduras/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/biossíntese , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Dicroísmo Circular , Microscopia Crioeletrônica , Citomegalovirus/genética , Regulação Fúngica da Expressão Gênica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Peptidil Transferases/química , Conformação Proteica , Aminoacil-RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Relação Estrutura-Atividade , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Leveduras/genética
20.
J Mol Biol ; 395(2): 417-29, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19900465

RESUMO

Guanosine 5'-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5'-monophosphate (adenyl-XMP) into guanosine 5'-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg(2+), ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.


Assuntos
Amidofosforribosiltransferase/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Pyrococcus horikoshii/enzimologia , Pirofosfatases/química , Amidofosforribosiltransferase/genética , Amidofosforribosiltransferase/metabolismo , Sequência de Aminoácidos , Amônia/farmacologia , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Pyrococcus horikoshii/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Especificidade por Substrato
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