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1.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31896597

RESUMO

TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.


Assuntos
Adenosina Trifosfatases/metabolismo , Nucleocapsídeo/metabolismo , Replicação Viral , Animais , Núcleo Celular/virologia , Citoplasma/virologia , DNA Helicases/metabolismo , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Células Sf9/virologia , Vacúolos/virologia , Proteínas Virais/metabolismo , Vírion
2.
Nat Commun ; 11(1): 162, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919357

RESUMO

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.


Assuntos
Antivirais/farmacologia , Hemaglutininas Virais/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/prevenção & controle , Peptídeos/farmacologia , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Autofagossomos/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Endossomos/metabolismo , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Células Sf9 , Spodoptera
3.
Insect Mol Biol ; 29(1): 92-103, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31456272

RESUMO

Ninety-four putative G protein-coupled receptors (GPCRs) were identified in the Musca domestica genome. They were annotated and compared with their homologues in Drosophila melanogaster. Phylogenetic analyses of the GPCRs from both species revealed that several family members shared a closer relationship based on the domain architecture. The expression profiles of these genes were examined by quantitative real-time PCR amongst three strains of the house fly, a near-isogenic line strain with imidacloprid resistance (N-IRS), the corresponding susceptible strain (CSS) and another strain derived from field populations with imidacloprid resistance (IRS). We found that five GPCR genes were upregulated in the N-IRS and eight GPCR genes were upregulated in the IRS strains compared to the CSS strain. The transgenic lines of D. melanogaster with the GPCR genes (LOC101899380 in the N-IRS strain and LOC101895664 in the IRS strain) exhibited significantly increased tolerance to imidacloprid, and higher expression of cytochrome P450 genes. Bioinformatic analysis of LOC101899380 was carried out based on its full-length nucleic acid sequence and putative amino acid sequence, and it was named Methuselah-like10 (Mthl10) owing to its homology with D. melanogaster Mthl10. A cell-base cell counting kit-8 toxicity assay demonstrated that the expression of the GPCR gene LOC101899380 in Spodoptera frugiperda (Sf9) cells using a baculovirus-mediated expression system can elevate the cell tolerance to imidacloprid, indirectly supporting the hypothesis that the GPCR gene LOC101899380 plays some role in imidacloprid resistance. These results should be useful for furthering understanding of the regulatory pathway by which house flies develop resistance.


Assuntos
Moscas Domésticas/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Receptores de Superfície Celular/genética , Animais , Animais Geneticamente Modificados , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Drosophila melanogaster/genética , Moscas Domésticas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Células Sf9
4.
Pan Afr Med J ; 33: 285, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31692877

RESUMO

Introduction: Pesticides are used as essential tools to control vector-borne diseases and agricultural pests and maintain quality and quantity crop production. Scientists attempt to use derived plant natural products due to environmental safety and low mammalian toxicity. Therefore, the cytotoxicity of malathion and Nepeta crispa essential oil against vertebrate L929 and invertebrate Sf9 cell lines were investigated. Methods: About 2×103 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates. Results: About 2×103 cells were placed into the wells of a 96-well plate experiments. Then appropriate concentrations of malathion and N. crispa essential oil added to the wells. The cells were allowed to grow for 3-5 days and estimated the cell numbers. Control cell wells contained only cells with DMSO. All treatments and controls repeated at least four replicates. Conclusion: Plant essential oil not only had no negative effects but also had boosting effects on the L929 cell viability. Nepeta crispa essential oil had negative effects on the Sf9 cell viability with the differences that derived plant natural products containing environmentally friendly and readily biodegradable derivatives, hydrolyzing rapidly in nature and nearly having no destructive effects on mammals and environment.


Assuntos
Malation/toxicidade , Nepeta/química , Óleos Voláteis/toxicidade , Praguicidas/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Óleos Voláteis/isolamento & purificação , Células Sf9 , Spodoptera
5.
Mol Cell ; 76(5): 784-796.e6, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31588022

RESUMO

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Mitocôndrias/enzimologia , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mitocondrial/metabolismo , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Animais , Biomarcadores Tumorais/genética , Exorribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mitocondrial/genética , Células Sf9 , Spodoptera
6.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505747

RESUMO

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.


Assuntos
Vacinas Bacterianas , Circovirus , Engenharia Genética , Mycoplasma hyopneumoniae , Vacinas Virais , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Circovirus/genética , Circovirus/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/farmacologia
7.
Nat Commun ; 10(1): 3956, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477691

RESUMO

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Lipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Anoctaminas/química , Anoctaminas/genética , Células COS , Cálcio/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera
8.
Mol Cell ; 76(1): 138-147.e5, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31473102

RESUMO

Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.


Assuntos
Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Humanos , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestrutura , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Ligação Proteica , Conformação Proteica , Proteólise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Relação Estrutura-Atividade
9.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484301

RESUMO

The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.


Assuntos
Resistência a Inseticidas/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , Animais , Culex , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Permetrina/farmacologia , Receptores Acoplados a Proteínas-G/genética , Células Sf9 , Transdução de Sinais/genética
10.
Nat Commun ; 10(1): 3740, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431622

RESUMO

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca2+. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca2+-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca2+ alone, and with both ADPR and Ca2+, resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Estrutura Quaternária de Proteína , Canais de Cátion TRPM/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Células HEK293 , Humanos , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Células Sf9 , Spodoptera , Canais de Cátion TRPM/química , Peixe-Zebra , Proteínas de Peixe-Zebra/química
11.
Nat Commun ; 10(1): 3814, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444342

RESUMO

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.


Assuntos
Complexo do Signalossomo COP9/ultraestrutura , Proteína NEDD8/ultraestrutura , Peptídeo Hidrolases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Complexo do Signalossomo COP9/isolamento & purificação , Complexo do Signalossomo COP9/metabolismo , Microscopia Crioeletrônica , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Proteína NEDD8/isolamento & purificação , Proteína NEDD8/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Ubiquitina-Proteína Ligases/isolamento & purificação , Ubiquitina-Proteína Ligases/metabolismo
12.
Biotechnol Lett ; 41(10): 1121-1131, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444662

RESUMO

OBJECTIVES: To analyze the effect of Ac25 on the proliferation of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) progeny virus and its function in virogenic stroma. RESULTS: AcMNPV is a model of baculovirus and is the most widely studied baculovirus. Ac25, as a single-stranded DNA-binding protein, is involved in viral genomic DNA replication. Viral proliferation assay showed that AcMNPV progeny virus could not be produced when Ac25 was knocked out, which indicated it was crucial for BV production. Absolute quantitative PCR analysis indicated that Ac25 was able to promote replication of the AcMNPV genome in host Sf9 cells. It was also found that Ac25 could increase the transcription level of 38k and vp39 late expression genes, and inhibit host cell proliferation. CONCLUSION: Ac25 is highly accumulated in the nucleus and promotes progeny virus production by stimulating viral genome replication and up-regulating the expression of late genes. Two potential applications of vAc-Ac25-EGFP were proposed: an improved bac-to-bac eukaryotic protein expression systems and biopesticides.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes Virais , /genética , Proteínas Virais/metabolismo , Liberação de Vírus , Replicação Viral , Animais , Proteínas de Ligação a DNA/genética , Células Sf9 , Spodoptera , Proteínas Virais/genética
13.
PLoS One ; 14(8): e0219475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31433806

RESUMO

Glycoprotein G (gG) is a conserved protein, and it has been described as a chemokine-binding protein in most members of the alphaherpesviruses. In case of the infectious laryngotracheitis virus (ILTV), an alphaherpesvirus that infects chickens, this protein is a virulence factor that plays an immunomodulatory role in the chicken immune response. Nevertheless, the gG production profile during ILTV infection has not yet been studied. In this study, we developed monoclonal antibodies in order to determine the gG production profile during ILTV infection in chicken hepatocellular carcinoma (LMH) cell cultures as well as embryonated specific-pathogen-free (SPF) chicken eggs and SPF chickens using a sandwich enzyme-linked immunosorbent assay (ELISA). Despite the fact that inoculated LMH cell cultures showed an increase in both gG production and viral genome copy number up to 96 h after inoculation, we observed that gG production started earlier than the increase in viral genome copy number in ILTV infected embryonated SPF chicken eggs. Likewise, a gG production peak and an increase of viral genome copy number was observed prior to the appearance of clinical signs in infected SPF chickens. According to the production profiles, gG was also produced quite early in eggs and chickens inoculated with ILTV. These findings contribute to the knowledge of the gG role during the ILTV infection as a virulence factor.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Galináceo 1/fisiologia , Proteínas do Envelope Viral/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Baculoviridae/genética , Galinhas/virologia , Genoma Viral/genética , Herpesvirus Galináceo 1/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
14.
J Microbiol Biotechnol ; 29(9): 1470-1477, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31434365

RESUMO

ß2-adrenergic receptor (ß2-AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ß-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 µg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 µg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ß2-AR would have great application potential in detection of ß-agonist residues.


Assuntos
Agonistas Adrenérgicos beta/análise , Técnicas Biossensoriais/métodos , Expressão Gênica , Receptores Adrenérgicos beta 2/metabolismo , Carne Vermelha/análise , Agonistas Adrenérgicos beta/metabolismo , Animais , Clonagem Molecular , Limite de Detecção , Ligação Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Células Sf9 , Spodoptera , Suínos
15.
Nat Commun ; 10(1): 3858, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451685

RESUMO

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.


Assuntos
Proteínas Oncogênicas/metabolismo , Óvulo/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Espermatozoides/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/isolamento & purificação , Oogênese , Ovário/citologia , Ovário/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Espermatogênese , Testículo/citologia , Testículo/patologia
16.
Genes Dev ; 33(19-20): 1397-1415, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31467087

RESUMO

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Animais , Linhagem Celular , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Infertilidade/genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Sequência , Células Sf9
17.
PLoS Genet ; 15(8): e1008331, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412019

RESUMO

Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.


Assuntos
Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/fisiologia , Receptores Dopaminérgicos/metabolismo , Animais , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Flupentixol/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/fisiologia , Muda/efeitos dos fármacos , Muda/fisiologia , Mariposas/efeitos dos fármacos , Interferência de RNA , Receptores Dopaminérgicos/genética , Células Sf9
18.
Insect Biochem Mol Biol ; 112: 103202, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31422153

RESUMO

The microRNA (miRNA) pathway is an epigenetic mechanism that plays important roles in various biological processes including host-virus interactions by regulating gene expression of the host and/or the virus. Previously, we showed that the cellular microRNAome in Spodoptera frugiperda (Sf9) cells is modulated following Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection suggesting that miRNAs may contribute in the cellular antiviral immunity. Here, we investigated the role of core components of the miRNA pathway in Sf9-AcMNPV interaction. Gene expression analyses showed that the expression levels of Dicer-1 (Dcr1), Argonaute-1 (Ago1) and Exportin-5 (Exp5) increased following AcMNPV infection particularly at 16 h post infection (hpi). Ran expression levels, however, decreased in response to virus infection. The expression levels of cellular miRNAs, miR-184 and let-7, also diminished at the post infection times further confirming differential expression of the cellular miRNAs following AcMNPV infection. To determine the role of the miRNA pathway in the interaction, we silenced key genes in the pathway using specific dsRNAs. RNAi of Dcr1, Ago1 and Ran enhanced viral DNA replication and reduced the abundance of miR-184 and let-7 underscoring the importance of the miRNA pathway in antiviral immunity in Sf9 cells. Suppression of the miRNA pathway in mock and infected cells had no effect on Ran expression levels suggesting miRNA-independent downregulation of this gene after virus infection. In conclusion, our results suggest the antiviral role of the miRNA pathway in Sf9 cells against AcMNPV. To modulate this immune response, AcMNPV represses host miRNAs likely through downregulation of Ran to enhance its replication in the host cells.


Assuntos
MicroRNAs/metabolismo , Spodoptera/imunologia , Spodoptera/virologia , Animais , DNA Viral , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Interferência de RNA , Células Sf9 , Spodoptera/genética , Replicação Viral
19.
Arch Insect Biochem Physiol ; 102(1): e21596, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31270854

RESUMO

ß-Asarone is the predominant component of the essential oil of rhizomes of Acorus calamus Linn ( Sweet flag). Although rhizome extracts from this plant have long been used for insect pest control, their cytotoxic effects on insect cells are not well understood. In this study, we evaluated the potency of ß-asarone as a natural insecticide by using a Spodoptera frugiperda cell line (Sf9). To assess the cytotoxic effects of ß-asarone on Sf9 cells, we observed morphologic changes in treated cells and performed a cell proliferation assay and a DNA fragmentation assay. After 24 and 48 h of treatment with ß-asarone, the proliferation of the Sf9 cells was inhibited in a dose-dependent manner, with IC50 values of 0.558 mg/ml at 24 h and 0.253 mg/ml at 48 h. Morphologic changes in ß-asarone-treated cells were typical of apoptosis and included loss of adhesion, cell shrinkage, and small apoptotic bodies. The DNA laddering present in ß-asarone-treated SF9 cells and annexin V assay confirmed that this compound can induce apoptosis in insect cells. Together, these findings suggest that apoptosis induction may be one mechanism through which ß-asarone inhibits the proliferation of insect cells and thus exerts insecticidal effects.


Assuntos
Anisóis/toxicidade , Acorus , Animais , Apoptose , Fragmentação do DNA , Células Sf9 , Spodoptera , Testes de Toxicidade
20.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261773

RESUMO

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.


Assuntos
Canal de Potássio ERG1/genética , Engenharia de Proteínas/métodos , Animais , Fracionamento Químico/métodos , Cristalografia por Raios X/métodos , Canal de Potássio ERG1/química , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Xenopus
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