Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.579
Filtrar
1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34204013

RESUMO

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Resistência à Doença/imunologia , Fusarium/fisiologia , Imunidade Vegetal , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Resistência à Doença/genética , Edição de Genes , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Vetores Genéticos/metabolismo , Lycopersicon esculentum/genética , Mutação/genética , Imunidade Vegetal/genética , Plasmídeos/genética
2.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elongação da Transcrição Genética
4.
SLAS Discov ; 26(6): 757-765, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1194439

RESUMO

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Assuntos
Adenosina/análogos & derivados , Ensaios de Triagem em Larga Escala , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/química , Adenosina/farmacologia , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metilação , Modelos Moleculares , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Trítio , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
5.
SLAS Discov ; 26(6): 766-774, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1192708

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Exonucleases/antagonistas & inibidores , Exorribonucleases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33921102

RESUMO

RRM1-an important DNA replication/repair enzyme-is the primary molecular gemcitabine (GEM) target. High RRM1-expression associates with gemcitabine-resistance in various cancers and RRM1 inhibition may provide novel cancer treatment approaches. Our study elucidates how RRM1 inhibition affects cancer cell proliferation and influences gemcitabine-resistant bladder cancer cells. Of nine bladder cancer cell lines investigated, two RRM1 highly expressed cells, 253J and RT112, were selected for further experimentation. An RRM1-targeting shRNA was cloned into adenoviral vector, Ad-shRRM1. Gene and protein expression were investigated using real-time PCR and western blotting. Cell proliferation rate and chemotherapeutic sensitivity to GEM were assessed by MTT assay. A human tumor xenograft model was prepared by implanting RRM1 highly expressed tumors, derived from RT112 cells, in nude mice. Infection with Ad-shRRM1 effectively downregulated RRM1 expression, significantly inhibiting cell growth in both RRM1 highly expressed tumor cells. In vivo, Ad-shRRM1 treatment had pronounced antitumor effects against RRM1 highly expressed tumor xenografts (p < 0.05). Moreover, combination of Ad-shRRM1 and GEM inhibited cell proliferation in both cell lines significantly more than either treatment individually. Cancer gene therapy using anti-RRM1 shRNA has pronounced antitumor effects against RRM1 highly expressed tumors, and RRM1 inhibition specifically increases bladder cancer cell GEM-sensitivity. Ad-shRRM1/GEM combination therapy may offer new treatment options for patients with GEM-resistant bladder tumors.


Assuntos
Adenoviridae/genética , Desoxicitidina/análogos & derivados , Técnicas de Silenciamento de Genes , Vetores Genéticos/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Ribonucleosídeo Difosfato Redutase/genética , Neoplasias da Bexiga Urinária/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Biomed Res Int ; 2021: 5512370, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33855069

RESUMO

Objective: This study is aimed at investigating the role of long noncoding RNA (lncRNA) RP11-815M8.1 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: RT-PCR was used to detect the expression of lncRNA RP11-815M8.1 before and after osteogenic differentiation of hBMSCs. The lncRNA RP11-815M8.1 in hBMSCs was overexpressed or silenced via lentiviral transfection. The transfection efficiency was detected by RT-PCR, and the proliferation of hBMSCs was determined by CCK-8. After 14 days of osteogenic differentiation of transfected hBMSCs, the expression of osteogenic transcription factors (ALP, OCN, OPN, Runx2, and Osterix) was detected by alizarin red staining and RT-PCR. The mRNAs directly regulated by lncRNA RP11-815M8.1 and targeted miRNAs were analyzed according to the positional relationship between lncRNA and mRNA in the genome and miRanda software. Results: The expression of lncRNA RP11-815M8.1 enhanced with increasing osteogenic differentiation time of hBMSCs. Two days after the transfection of hBMSCs, lncRNA RP11-815M8.1 expression was significantly increased in the overexpression group and significantly decreased in the knockdown group, compared to control cells. The CCK-8 assay showed that overexpression and knockdown of lncRNA RP11-815M8.1 did not affect the proliferation of hBMSCs. After 14 days of differentiation of hBMSCs, stronger alizarin red staining was observed in the overexpression groups, and the expression of osteogenic transcription factors was increased in the overexpression group compared to the control. In the knockdown group, alizarin red staining and the expression of osteogenic transcription factors were decreased. Bioinformatics analysis showed that lncRNA RP11-815M8.1 was directly associated with one mRNA, 27 interacting miRNAs, and 20 miRNA-targeted mRNAs. Conclusion: The osteogenic differentiation of hBMSCs can be promoted by lncRNA RP11-815M8.1 in vitro.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , RNA Longo não Codificante/metabolismo , Proliferação de Células/genética , Regulação da Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
8.
Mol Cell ; 81(11): 2403-2416.e5, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33852892

RESUMO

The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular/química , Fator de Iniciação 4E em Eucariotos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Proteína Regulatória Associada a mTOR/química , Serina-Treonina Quinases TOR/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaetomium/química , Chaetomium/genética , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Especificidade por Substrato , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
9.
SLAS Discov ; 26(6): 766-774, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33870746

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Exonucleases/antagonistas & inibidores , Exorribonucleases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
10.
SLAS Discov ; 26(6): 757-765, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33874769

RESUMO

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Assuntos
Adenosina/análogos & derivados , Ensaios de Triagem em Larga Escala , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/química , Adenosina/farmacologia , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metilação , Modelos Moleculares , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Trítio , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
11.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799598

RESUMO

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.


Assuntos
Aflatoxina B1/toxicidade , Efeito Fundador , Medições Luminescentes/métodos , Primaquina/toxicidade , Imagem com Lapso de Tempo/métodos , Xenobióticos/toxicidade , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentração Inibidora 50 , Luciferases/genética , Luciferases/metabolismo , Luminescência , Camundongos
12.
SLAS Discov ; 26(6): 749-756, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1136206

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Assuntos
Antivirais/farmacologia , Exorribonucleases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Nitrocompostos/farmacologia , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antiparasitários/química , Antiparasitários/farmacologia , Antivirais/química , COVID-19/virologia , Clonagem Molecular , Reposicionamento de Medicamentos , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Espectrometria de Massas/métodos , Metilação , Nitrocompostos/química , Medicamentos sob Prescrição/química , Medicamentos sob Prescrição/farmacologia , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazóis/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
13.
Mol Biol Rep ; 48(3): 2053-2061, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33660095

RESUMO

Red rot caused by Colletotrichum falcatum, is one of the economically important disease of sugarcane and breeding for resistant varieties is considered to be the major solution to manage the disease. However, breakdown of red rot resistance become usual phenomenon due to development of newer races by culture adaptation on newly released varieties. Hence it is needed to characterize the genes responsible for pathogen virulence in order to take care of host resistance or to manage the disease by other methods. The transcript studies gave foundation to characterize the huge number of pathogenicity determinants and their role in pathogenesis. Here we studied role of two important genes viz., Glucose Transporter (GT) and Sucrose Non-Fermenting1 (SNF1) during pathogenesis of C. falcatum, which said to be involved in carbon source metabolism. Sugar metabolism has a vital role in disease progression of C. falcatum by regulating their cell growth, metabolism and development of the pathogen during various stages of infection. The present study was aimed to find out the role of GT and SNF1 genes in response to pathogenicity by RNA silencing (RNAi) approach. Knock-down of the target pathogenicity gene homologs in standard C. falcatum isolate Cf671 was carried out by amplifying sense and antisense fragments of targets individually using pSilent-1 vector. The expression cassette was cloned into the binary vector pCAMBIA1300 followed by fungal transformation through Agarobacterium mediated transformation. Resulted mutants of both the genes showed less virulence compared to wild type isolate. Simultaneously, both the mutants did not produce spores. Moreover, the molecular confirmation of the mutants displayed the expression of hygromycin gene with reduced expression of the target gene during host-pathogen interaction. Knockdown of the pathogenicity related genes (GT and SNF1) by RNAi approach corroborate the possible role of the genes in causing the disease.


Assuntos
Colletotrichum/genética , Fermentação , Técnicas de Silenciamento de Genes , Genes Fúngicos , Proteínas Facilitadoras de Transporte de Glucose/genética , Doenças das Plantas/microbiologia , Saccharum/microbiologia , Sacarose/metabolismo , Agrobacterium/metabolismo , Cinamatos/metabolismo , Colletotrichum/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Vetores Genéticos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Higromicina B/análogos & derivados , Higromicina B/metabolismo , Mutação/genética , Fenótipo , Transformação Genética
14.
J Mol Biol ; 433(10): 166924, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33711344

RESUMO

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.


Assuntos
Apoproteínas/química , Proteínas de Transporte/química , Proteínas Ferro-Enxofre/química , Ferro/química , Mitocôndrias/química , Proteínas Mitocondriais/química , Enxofre/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Enxofre/metabolismo
15.
J Mol Biol ; 433(10): 166947, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744315

RESUMO

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.


Assuntos
GMP Cíclico/química , Guanilato Ciclase/química , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Segmento Externo da Célula Bastonete/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Reagentes para Ligações Cruzadas/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Succinimidas/química
16.
J Mol Biol ; 433(10): 166948, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744316

RESUMO

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43's NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Intrinsicamente Desordenadas/química , Coloração e Rotulagem/métodos , Sítios de Ligação , Cumarínicos/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química
18.
Science ; 372(6541)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33766941

RESUMO

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8+ T cell priming, resulting in CD8+ T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/metabolismo , Vetores Genéticos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Vacinas contra a SAIDS/imunologia , Animais , Linhagem Celular , Citomegalovirus/genética , Epitopos de Linfócito T/imunologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/genética , Ligantes , Macaca mulatta , Fragmentos de Peptídeos/genética , Transporte Proteico , Vírus da Imunodeficiência Símia
19.
Nat Commun ; 12(1): 1950, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782388

RESUMO

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.


Assuntos
Antígenos CD4/química , Guanidinas/química , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Indenos/química , Receptores Virais/química , Animais , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Antígenos CD4/antagonistas & inibidores , Antígenos CD4/genética , Antígenos CD4/metabolismo , Células CHO , Clonagem Molecular , Cricetulus , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanidinas/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Indenos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
20.
Nat Chem Biol ; 17(5): 608-614, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33686294

RESUMO

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.


Assuntos
Crioprotetores/química , Hidrogéis/química , Proteína FUS de Ligação a RNA/química , Sefarose/química , Materiais Biomiméticos/química , Clonagem Molecular , Citoesqueleto/química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...