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1.
Food Chem ; 329: 127146, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32526599

RESUMO

A non-target screening method of cyclopeptide toxins and their analogues in mushroom was developed, using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) followed by mass spectrometry databases retrieval and software tools analysis for the candidate analogues. Three cyclopeptide toxins in the toxic mushroom Amanita rimosa were firstly screened without standard, and two of them were unknown analogues which were tentatively identified by the accurate masses, isotopic patterns and characteristic fragments. A validated quantitative method was performed to rapidly quantify three major cyclopeptide toxins in the Amanita rimosa sample including α-manitin, ß-amanitin and phalloidin, and their contents were detected to be 4.52 mg/kg, 2.37 mg/kg and 2.53 mg/kg, respectively. The developed method has good selectivity and sensitivity for rapid and comprehensive screening the cyclopeptide toxins and their analogues in mushrooms at trace levels. Successful non-target screening of trace cyclopeptide toxin analogues will guarantee the food safety in mushrooms consumption.


Assuntos
Alfa-Amanitina/química , Amanita/química , Amanitinas/química , Faloidina/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas
2.
Toxicon ; 183: 61-68, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32473253

RESUMO

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding ß-amanitin (ß-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization.


Assuntos
Amanita/genética , Peptídeos Cíclicos/genética , Alfa-Amanitina , Amanitinas , Sequência de Aminoácidos , China , Perfilação da Expressão Gênica , Alinhamento de Sequência , Toxinas Biológicas , Transcriptoma
3.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941775

RESUMO

Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/ß). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response.IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Elonguina/metabolismo , Vírus La Crosse/enzimologia , Proteínas não Estruturais Virais/metabolismo , Células A549 , Alfa-Amanitina/metabolismo , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Interferons/metabolismo , Vírus La Crosse/genética , Fenótipo , RNA Interferente Pequeno/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Transcrição Genética , Células Vero , Fatores de Virulência/metabolismo
4.
Anal Chim Acta ; 1093: 142-149, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31735207

RESUMO

α-amanitin is the most toxic amanita in mushrooms with lethal dose to humans around 0.1 mg. Kg-1. Hence, early identification of the poison would improve survival rates and prevent lethal poisoning cases. In this study, molecularly imprinted photonic crystal (MIPC) sensor was prepared by combining molecular imprinting with photonic crystal templates and tested towards the detection of α-amanitin. In this process, synthesized moiety of α-amanitin was utilized as template, dispersed SiO2 colloidal photonic crystal as carrier, methacrylic acid (MAA) as functional monomer, and ethylene glycol dimethacrylate (EDGMA) as crosslinker. The adsorption behavior of MIPC towards α-amanitin in ethanol solution showed shifts in diffraction peaks of MIPC upon binding with α-amanitin molecules. The reflection peak wavelength varied linearly with α-amanitin concentration according to the correlation formula: λ = 15.417c+489.17 (R2 = 0.9985). The recognition process was accompanied by gradual color change in MIPC film. The prepared MIPC sensor possessed wide linear range (10-9-10-3 mg L-1), change in visual color, low detection limit (10-10 mg L-1), short response time (2 min), and good reusability. The MIPC film was then tested towards the detection of α-amanitin in real biological samples (mushroom, urine, and serum) and showed reasonable shift in diffraction peaks and color change upon soaking in solutions spiked with α-amanitin at 10-6 mg L-1 and 10-3 mg L-1, suggesting the suitability of the film for the rapid identification of α-amanitin in complex sample matrices. Overall, the proposed sensor looks promising for the rapid identification of α-amanitin in clinical analysis and food poisoning.


Assuntos
Alfa-Amanitina/análise , Colorimetria/métodos , Dióxido de Silício/química , Agaricales/química , Alfa-Amanitina/sangue , Alfa-Amanitina/urina , Feminino , Humanos , Limite de Detecção , Impressão Molecular/métodos , Ácidos Polimetacrílicos/química , Reprodutibilidade dos Testes
5.
Plant Cell Physiol ; 60(11): 2584-2596, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373371

RESUMO

During seed germination, proteins are translated not only from mRNAs newly transcribed upon imbibition but also from long-lived mRNAs that are synthesized during seed maturation and stored in the mature dry seeds. To clarify the distinct roles of proteins translated from long-lived mRNAs and de novo transcribed mRNAs in germinating rice embryos, proteome analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining the use of a transcriptional inhibitor was performed. We observed that α-amanitin significantly represses transcription in germinating embryos; nevertheless, the embryos could germinate, albeit slowly. The proteomic analysis revealed that a total of 109 proteins were translated from long-lived mRNAs associated with germination as well as 222 proteins whose expression were dependent on de novo transcription upon imbibition. Transcriptomic datasets available in public databases demonstrated that mRNAs of the 222 proteins notably increased during germination while those of the 109 proteins highly accumulated in dry embryos and constitutively expressed upon imbibition. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many of the 109 proteins from long-lived mRNAs are implicated in energy production such as glycolysis or annotated as nucleotide binding proteins, while the 222 proteins are involved in pathways such as pyruvate metabolism and TCA cycle following glycolysis, and momilactones biosynthesis. We propose that long-lived mRNAs support initial energy production and activation of translational machinery upon imbibition whereas de novo transcription accelerates the energy production after glycolysis, which enables rice seeds to germinate vigorously.


Assuntos
Oryza/metabolismo , RNA Mensageiro/metabolismo , Sementes/metabolismo , Alfa-Amanitina/metabolismo , Germinação/fisiologia , Proteômica
6.
Food Chem ; 298: 125031, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260975

RESUMO

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita ß-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas.


Assuntos
Amanitinas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Agaricales/genética , Alfa-Amanitina/genética , Amanita/genética , Limite de Detecção , Intoxicação Alimentar por Cogumelos/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
7.
Toxicon ; 156: 34-40, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30399359

RESUMO

Amanitin-induced apoptosis is proposed to have a significant effect on the pathogenesis of liver damage. However, few reports have focused on proteome changes induced by α-amanitin (α-AMA). Here, we evaluated changes in mitochondrial proteins of hepatocytes in response to 2 µM α-AMA, a concentration at which α-AMA-induced cell damage could be rescued at cellular level by common clinical drugs. We found 56 proteins were differentially expressed in an α-AMA-treated group. Among them, 38 proteins were downregulated and 18 were upregulated. Downregulated functional proteins included importer TOMM40, respiratory chain component cytochrome C, and metabolic enzymes of citrate acid cycle such as malate dehydrogenase, which localize on the mitochondrial outer membrane, inner membrane and matrix respectively. Immunoblot analysis showed that α-AMA decreased mitochondrial import receptor subunit TOMM40 and cytochrome c accompanied by an increase in the cytosol although their total protein levels were not affected significantly. The mitochondrial membrane potential was also destroyed by α-AMA and was restored by the clinical drug silibinin. Immunofluorescence suggested that mitochondrial morphology did not change. Taken together, our results provide further insights into the toxic mechanism of α-AMA on hepatocytes.


Assuntos
Alfa-Amanitina/toxicidade , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Hepatócitos/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo
8.
Mol Cell ; 72(5): 888-901.e7, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30344095

RESUMO

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Fatores de Elongação da Transcrição/genética , Alfa-Amanitina/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Morfolinas/farmacologia , Células NIH 3T3 , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Venenos/farmacologia , Pirimidinas/farmacologia , Pironas/farmacologia , Transdução de Sinais , Fatores de Elongação da Transcrição/metabolismo
9.
World J Gastroenterol ; 24(34): 3834-3848, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30228778

RESUMO

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Imunoconjugados/farmacologia , Evasão Tumoral/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Alfa-Amanitina/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Receptores Coestimuladores e Inibidores de Linfócitos T/antagonistas & inibidores , Receptores Coestimuladores e Inibidores de Linfócitos T/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Imunoconjugados/uso terapêutico , Imunoterapia/métodos , Instabilidade de Microssatélites/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , RNA Polimerase II/antagonistas & inibidores , Resultado do Tratamento , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genética
10.
Syst Biol Reprod Med ; 64(5): 389-398, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30136857

RESUMO

The episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression. ABBREVIATIONS: ANOVA: analysis of the variance; DMEM: Dulbecco's modified Eagle's medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Receptores LHRH/metabolismo , Alfa-Amanitina/farmacologia , Animais , Linhagem Celular Transformada , Meios de Cultura , Ciclofosfamida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hormônio Liberador de Gonadotropina/biossíntese , Hormônio Liberador de Gonadotropina/metabolismo , Camundongos , Plasmídeos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/genética , Transfecção
11.
Chemistry ; 24(68): 17869-17880, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29987917

RESUMO

The development of synthetic methods to prepare conformationally constrained peptides and peptide-polyketide hybrids remain an important chemical challenge. It is known that structural rigidity correlates with the specificity, bioactivity, and stability of these peptide systems, thus rigid systems are particularly attractive leads for development of potent biopharmaceuticals. Herein we provide an overview of recent developments in the syntheses of naturally derived constrained peptides and peptide-polyketide hybrids, with a particular emphasis on those systems containing an ene-like bond.


Assuntos
Produtos Biológicos/síntese química , Peptídeos Cíclicos/síntese química , Policetídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Alcaloides/síntese química , Alcaloides/química , Alfa-Amanitina/síntese química , Alfa-Amanitina/química , Sequência de Aminoácidos , Aminoácidos/síntese química , Aminoácidos/química , Produtos Biológicos/química , Iminas/síntese química , Iminas/química , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Conformação Molecular , Peptídeos Cíclicos/química , Policetídeos/química
12.
Int J Mol Sci ; 19(7)2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029518

RESUMO

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.


Assuntos
Alfa-Amanitina/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Alfa-Amanitina/química , Animais , Linhagem Celular Tumoral , Endocitose , Fator 2 de Crescimento de Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Oligopeptídeos/química , Estrutura Secundária de Proteína , Transdução de Sinais
13.
Gene ; 662: 123-130, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29627524

RESUMO

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis.


Assuntos
Alfa-Amanitina/genética , Amanita/genética , Serina Endopeptidases/genética , Alfa-Amanitina/biossíntese , Alfa-Amanitina/metabolismo , Amanitinas/genética , Amanitinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases/genética , Clonagem Molecular/métodos , Carpóforos/genética , Regulação Fúngica da Expressão Gênica/genética , Peptídeos Cíclicos/genética , Filogenia , Serina Endopeptidases/metabolismo , Toxinas Biológicas/metabolismo
14.
J Biol Chem ; 293(19): 7189-7194, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29550768

RESUMO

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.


Assuntos
Alfa-Amanitina/química , Inibidores Enzimáticos/química , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/química , Elongação da Transcrição Genética/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio , Conformação Proteica , Homologia de Sequência de Aminoácidos , Suínos
15.
J Am Chem Soc ; 140(21): 6513-6517, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29561592

RESUMO

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2 S,3 R,4 R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.


Assuntos
Agaricales/química , Alfa-Amanitina/síntese química , Micotoxinas/síntese química , Alfa-Amanitina/química , Alfa-Amanitina/farmacologia , Animais , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Modelos Moleculares , Conformação Molecular , Micotoxinas/química , Micotoxinas/farmacologia , Relação Estrutura-Atividade
16.
Basic Clin Pharmacol Toxicol ; 122(6): 633-642, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29285878

RESUMO

Amanita phalloides species mushrooms containing alpha-amanitin (α-AMA) are responsible for the majority of fatal mushroom intoxications and can lead to severe poisonings resulting in hepatotoxicity and acute hepatic failure. Existing antidotes, such as silibinin, are not sufficiently effective in the prevention and/or resolution of α-AMA-induced hepatotoxicity. We investigated the effects of resveratrol on α-AMA-induced hepatotoxicity and compared with silibinin, a known antidote using in vivo and in vitro toxicity models. In the in vivo protocol, resveratrol (30 mg/kg) was given simultaneously with α-AMA (α-AMA + SR) or 12 (α-AMA + 12R) or 24 (α-AMA + 24R) hr after α-AMA administration. Silibinin (5 mg/kg) (α-AMA + Sil) and normal saline (α-AMA + NS) were given simultaneously with α-AMA. We found that liver transaminase levels in α-AMA + SR and α-AMA + 12R groups and histomorphologic injury score in the α-AMA + SR, α-AMA + 12R, α-AMA + 24R and α-AMA + Sil groups were significantly lower than that of the α-AMA + NS group. Resveratrol decreased mononuclear cell infiltration, necrosis and active caspase-3 immunopositivity in the liver. In the in vitro protocol, the effects of resveratrol and silibinin were evaluated in a reduction in cell viability induced by α-AMA in THLE-2 and THLE-3 hepatocytes. Neither resveratrol nor silibinin was found to be effective in increasing cell viability decreased by α-AMA + NS. As a conclusion, resveratrol was found to be effective in α-AMA-induced hepatotoxicity with its anti-inflammatory properties in in vivo conditions. It is a promising compound with the potential for use in the treatment of hepatotoxicity associated with Amanita phalloides type mushroom poisonings.


Assuntos
Alfa-Amanitina/antagonistas & inibidores , Alfa-Amanitina/toxicidade , Antioxidantes/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Intoxicação Alimentar por Cogumelos/tratamento farmacológico , Inibidores da Síntese de Ácido Nucleico/toxicidade , Substâncias Protetoras/uso terapêutico , Silimarina/uso terapêutico , Estilbenos/uso terapêutico , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Humanos , Fígado/enzimologia , Fígado/patologia , Resveratrol , Silibina
17.
Sci Rep ; 7(1): 15763, 2017 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-29150675

RESUMO

To study the relationship between chromatin condensation, gene transcription and developmental competence during oocyte maturation and to explore the mechanisms by which meiotic arrest maintenance (MAM) and sexual maturity improve oocyte competence, we examined effects of MAM with roscovitine or db-cAMP on chromatin condensation, gene transcription and developmental potential of NSN or SN oocytes from prepubertal or adult mice. MAM with roscovitine improved the developmental competence and global gene transcription of prepubertal NSN (prep-NSN) and adult-SN oocytes while having no effect on those of prep-SN oocytes. MAM with db-cAMP facilitated neither development nor transcription in any type of oocytes. MAM with either roscovitine or db-cAMP promoted chromatin condensation of prep-NSN oocytes. MAM with roscovitine promoted gene transcription and chromatin condensation simultaneously through inhibiting cyclin-dependent kinase (CDK) 5 and 2, respectively. The results suggested that MAM with roscovitine improved oocyte competence by promoting gene transcription via inhibiting CDK5. Oocyte cytoplasmic maturation is correlated with gene transcription but not with chromatin condensation. The difference in developmental competence between prepubertal NSN and SN oocytes and between prepubertal and adult SN oocytes was because while the former had not, the latter had completed or acquired the ability for transcription of important genes.


Assuntos
Pontos de Checagem do Ciclo Celular , Meiose , Oócitos/citologia , Maturidade Sexual , Alfa-Amanitina/farmacologia , Animais , Bucladesina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Isoquinolinas/farmacologia , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , Roscovitina/farmacologia , Maturidade Sexual/efeitos dos fármacos , Sulfonamidas/farmacologia , Transcrição Genética/efeitos dos fármacos
18.
Nucleic Acids Res ; 45(22): 12715-12722, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29036442

RESUMO

H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Histonas/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Alfa-Amanitina/farmacologia , Flavonoides/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histonas/metabolismo , Humanos , Modelos Genéticos , Nucleossomos/genética , Nucleossomos/metabolismo , Piperidinas/farmacologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-28898719

RESUMO

α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.


Assuntos
Alfa-Amanitina/sangue , Alfa-Amanitina/toxicidade , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Alfa-Amanitina/química , Alfa-Amanitina/farmacocinética , Animais , Cromatografia Líquida/economia , Estabilidade de Medicamentos , Limite de Detecção , Modelos Lineares , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/economia , Toxicocinética
20.
J Neurosci ; 37(40): 9675-9685, 2017 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-28887385

RESUMO

Reactivated memories can be modified during reconsolidation, making this process a potential therapeutic target for posttraumatic stress disorder (PTSD), a mental illness characterized by the recurring avoidance of situations that evoke trauma-related fears. However, avoidance memory reconsolidation depends on a set of still loosely defined boundary conditions, limiting the translational value of basic research. In particular, the involvement of the hippocampus in fear-motivated avoidance memory reconsolidation remains controversial. Combining behavioral and electrophysiological analyses in male Wistar rats, we found that previous learning of relevant nonaversive information is essential to elicit the participation of the hippocampus in avoidance memory reconsolidation, which is associated with an increase in theta- and gamma-oscillation power and cross-frequency coupling in dorsal CA1 during reactivation of the avoidance response. Our results indicate that the hippocampus is involved in memory reconsolidation only when reactivation results in contradictory representations regarding the consequences of avoidance and suggest that robust nesting of hippocampal theta-gamma rhythms at the time of retrieval is a specific reconsolidation marker.SIGNIFICANCE STATEMENT Posttraumatic stress disorder (PTSD) is characterized by maladaptive avoidance responses to stimuli or behaviors that represent or bear resemblance to some aspect of a traumatic experience. Disruption of reconsolidation, the process by which reactivated memories become susceptible to modifications, is a promising approach for treating PTSD patients. However, much of what is known about fear-motivated avoidance memory reconsolidation derives from studies based on fear conditioning instead of avoidance-learning paradigms. Using a step-down inhibitory avoidance task in rats, we found that the hippocampus is involved in memory reconsolidation only when the animals acquired the avoidance response in an environment that they had previously learned as safe and showed that increased theta- and gamma-oscillation coupling during reactivation is an electrophysiological signature of this process.


Assuntos
Aprendizagem da Esquiva/fisiologia , Hipocampo/fisiologia , Consolidação da Memória/fisiologia , Alfa-Amanitina/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Ondas Encefálicas/efeitos dos fármacos , Ondas Encefálicas/fisiologia , Hipocampo/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Consolidação da Memória/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ratos , Ratos Wistar
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