RESUMO
A topologically complex peptide [4]catenane with the crossing number of 12 was synthesized by a folding and assembly strategy wherein the folding and metal-directed self-assembly of a short peptide fragment occur simultaneously. The latent Ω-looped conformation of the Pro-Gly-Pro sequence was found only when pyridines at the C- and N-termini coordinatively bind metal ions (Ag(I) or Au(I) ). Crystallographic studies revealed that the Ω-looped motifs formed four M3 L3 macrocycles that were intermolecularly entwined to generate an unprecedented peptide [4]catenane topology.
Assuntos
Antracenos/química , Peptídeos/química , Ressonância Magnética Nuclear Biomolecular , Dobramento de ProteínaRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by ß-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Humulus/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Óleos Voláteis/química , Sesquiterpenos de Eudesmano/química , Sesquiterpenos de Eudesmano/farmacologia , Sesquiterpenos/química , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Humanos , Canal de Cátion TRPA1RESUMO
Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/biossíntese , Anticolesterolemiantes/farmacologia , Colesterol/metabolismo , Receptores Nucleares Órfãos/agonistas , Quercetina/análogos & derivados , Animais , Arteriosclerose/prevenção & controle , Transporte Biológico/efeitos dos fármacos , Flavonoides/análise , Flavonoides/farmacologia , Ligantes , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Nelumbo/química , Receptores Nucleares Órfãos/metabolismo , Extratos Vegetais/farmacologia , Quercetina/metabolismo , Quercetina/farmacologiaRESUMO
Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 µg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.
Assuntos
Alquil e Aril Transferases/isolamento & purificação , Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/isolamento & purificação , Dimetilaliltranstransferase/metabolismo , Lithospermum/enzimologia , Animais , Dimetilaliltranstransferase/genética , Cinética , Lithospermum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Especificidade por SubstratoRESUMO
Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.
Assuntos
Dimetilaliltranstransferase/metabolismo , Glycine max/enzimologia , Lotus/enzimologia , Engenharia Metabólica/métodos , Plantas Geneticamente Modificadas/enzimologia , Polifenóis/biossíntese , Dimetilaliltranstransferase/genética , Flavanonas/administração & dosagem , Lotus/genética , Plantas Geneticamente Modificadas/genética , Prenilação/genética , Sophora/enzimologia , Sophora/genética , Glycine max/genética , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por SubstratoRESUMO
The complexity and heterogeneity of tumours have hindered efforts to identify commonalities among different cancers. Furthermore, because we have limited information on the prevalence and nature of ubiquitous molecular events that occur in neoplasms, it is unfeasible to implement molecular-targeted cancer screening and prevention. Here, we found that the FEAT protein is overexpressed in most human cancers, but weakly expressed in normal tissues including the testis, brain, and liver. Transgenic mice that ectopically expressed FEAT in the thymus, spleen, liver, and lung spontaneously developed invasive malignant lymphoma (48%, 19/40) and lung-metastasizing liver cancer (hepatocellular carcinoma) (35%, 14/40) that models human hepatocarcinogenesis, indicating the FEAT protein potently drives tumorigenesis in vivo. Gene expression profiling suggested that FEAT drives receptor tyrosine kinase and hedgehog signalling pathways. These findings demonstrate that integrated efforts to identify FEAT-like ubiquitous oncoproteins are useful and may provide promising approaches for cost-effective cancer screening and prevention.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Metiltransferases/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Regulação para CimaRESUMO
The monoterpene D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia). Apoptosis was not observed in the limonene-producing cells. A concomitant increase in the level of apoptosis-related protein Bcl-2 (B-cell lymphoma protein 2) and decreases in the levels of Bad (Bcl-2 antagonist of cell death) and phosphorylated JNK (c-Jun N-terminal kinase) were observed in limonene-producing cells. Limonene-producing cells may provide a useful new system to investigate the in vivo function of this monoterpene.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cicloexenos/metabolismo , Liases Intramoleculares/metabolismo , Lamiaceae/enzimologia , Terpenos/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Ciclo Celular , Proliferação de Células , Clonagem Molecular , Citometria de Fluxo , Cromatografia Gasosa-Espectrometria de Massas , Liases Intramoleculares/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Limoneno , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Plasmídeos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K (m) values for geranyl diphosphate and PHB were 59.7 and 6.04 microM, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa.
Assuntos
Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Ubiquinona/biossíntese , Alquil e Aril Transferases/análise , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Sequência de Aminoácidos , DNA de Plantas/genética , DNA de Plantas/fisiologia , Éxons/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Oryza/química , Parabenos/metabolismo , Proteínas de Plantas/análise , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por SubstratoRESUMO
4-Hydroxybenzoate polyprenyl diphosphate transferase (4HPT) is the key enzyme that transfers the prenyl side chain to the benzoquione frame in ubiquinone (UQ) biosynthesis. The Arabidopsis AtPPT1 cDNA encoding 4HPT was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on the information of the Arabidopsis genomic sequence, and the function of the gene was determined. Heterologous expression of the AtPPT1 gene enabled restoration of the respiratory ability and UQ synthesis in a yeast mutant that was defective in 4HPT activity. The mitochondrial fraction that was prepared from the yeast mutant, which expressed the AtPPT1 gene, exhibited 4HPT enzymatic activity with geranyl diphosphate (GPP) as the prenyl substrate. This indicated that the AtPPT1 gene encodes active 4HPT with a broad substrate specificity in terms of the prenyl donor. The AtPPT1 mRNA was predominantly expressed in the flower cluster, and the green fluorescent protein (GFP) fused with the signal peptide of AtPPT1 was translocated into the mitochondria. T-DNA insertion mutation that disrupts the AtPPT1 gene in Arabidopsis resulted in the arrest of embryo development at an early stage of zygotic embryogenesis. These results demonstrate that the AtPPT1 gene involved in the biosynthesis of mitochondrial UQ plays an essential role in embryo development in Arabidopsis .
Assuntos
Alquil e Aril Transferases/genética , Arabidopsis/genética , Sementes/genética , Ubiquinona/biossíntese , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/metabolismo , Northern Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Ubiquinone (UQ), an electron carrier in the respiratory chain ranging from bacteria to humans, shows antioxidative activity in vitro, but its physiological role in vivo is not yet clarified in plants. UQ biosynthesis was modified by overexpressing the yeast gene coq2, which encodes p-hydroxybenzoate:polyprenyltransferase, to increase the accumulation of UQ-6 in yeast and UQ-10 in tobacco. The yeast and tobacco transgenic lines showed about a three- and six-fold increase in UQ, respectively. COQ2 polypeptide, the localization of which was forcibly altered to the endoplasmic reticulum, had the same or a greater effect as mitochondria-localized COQ2 on the increase in UQ in both the yeast and tobacco transformants, indicating that the UQ intermediate is transported from the endoplasmic reticulum to the mitochondria. Plants with a high UQ level are more resistant to oxidative stresses caused by methyl viologen or high salinity. This is attributable to the greater radical scavenging ability of the transgenic lines when compared with the wild type.
Assuntos
Alquil e Aril Transferases/genética , Nicotiana/genética , Saccharomyces cerevisiae/genética , Ubiquinona/biossíntese , Alquil e Aril Transferases/metabolismo , Retículo Endoplasmático/enzimologia , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Estresse Oxidativo , Fenótipo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio , Nicotiana/metabolismo , Ubiquinona/genéticaRESUMO
Limonene synthase (LS) catalyses the stereo-specific cyclization of geranyl diphosphate (GPP) to form a monocyclic monoterpene, limonene. In an attempt to engineer monoterpene biosynthesis, three expression constructs of LS cDNA of Perilla frutescens, which were designed to be localized in either the plastid, the cytosol or the endoplasmic reticulum (ER), were introduced into tobacco in order to examine differences in enzyme activity and the productivity of limonene. High and moderate enzyme activity, respectively, was observed for plastid- and cytosol-localized LS, whereas no enzyme activity was seen for ER-localized LS, suggesting that the plastid is the preferred compartment for LS, while LS may also have an active form in the cytosol. The formation of limonene in vivo was confirmed by gas chromatography-mass spectrometry (GC-MS) in leaf extracts of both plastid- and cytosol-localized LS transgenic plants. The amount of limonene in plastid-localized LS transgenic plants was 143 ng g-1 fresh wt, whereas that in the cytosol-type was 40 ng g-1 fresh wt, and these limonene contents increased by 2.7-fold and 3.0-fold, respectively, with the addition of methyl jasmonate. The headspace analyses showed that the plastid- and the cytosol-localized LS transgenic plants (12 cm high) emitted 390 ng and 515 ng limonene per month, respectively. The possibility of genetically engineering monoterpene production is discussed.