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1.
Rev Bras Parasitol Vet ; 29(3): e005820, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756774

RESUMO

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.


Assuntos
Proteínas de Bactérias , Doenças do Cão , Ehrlichia canis , Ehrlichiose/veterinária , Proteínas Recombinantes , Testes Sorológicos/veterinária , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brasil , Linhagem Celular , Doenças do Cão/diagnóstico , Cães , Ehrlichia canis/genética , Ehrlichiose/diagnóstico , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
2.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848127

RESUMO

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Dobramento de Proteína , Modificação Traducional de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Supressão Genética , Temperatura
3.
Nat Commun ; 11(1): 4263, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848132

RESUMO

Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação , Domínio AAA , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hidrólise , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Nat Commun ; 11(1): 3834, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737309

RESUMO

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.


Assuntos
Escherichia coli/efeitos dos fármacos , Optogenética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Tetraciclina/farmacologia , Tetraciclinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios Ultravioleta
5.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
6.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , 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 , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
7.
Appl Microbiol Biotechnol ; 104(19): 8427-8437, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813067

RESUMO

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Proteínas Virais/genética , Animais , Embrião de Galinha , Galinhas , Infecções por Coronavirus/veterinária , DNA Complementar , Recombinação Homóloga , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
8.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1305-1313, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748588

RESUMO

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA257₋264 peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLP(OVA)) in vitro after purification. The VLP(OVA) was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA257₋264 peptide into VP3 and its location. The results show that insertion of OVA257₋264 into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLP(OVA) in size was larger than VLPs, and the OVA257₋264 peptide was located on the surface of VLP(OVA).


Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas de Partículas Semelhantes a Vírus , Animais , Escherichia coli/genética , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Biochem Biophys Res Commun ; 529(2): 257-262, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32703420

RESUMO

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.


Assuntos
Bombyx/citologia , Bombyx/virologia , Nucleopoliedrovírus/genética , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Animais , Bombyx/enzimologia , Linhagem Celular , Clonagem Molecular , Furina/metabolismo , Nucleopoliedrovírus/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
10.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
11.
Mol Cell ; 79(3): 390-405.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32619402

RESUMO

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.


Assuntos
Moléculas de Adesão Celular/química , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores da Família Eph/antagonistas & inibidores , Receptores da Família Eph/química , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato
12.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32647346

RESUMO

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Peptidil Dipeptidase A/química , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Quirópteros/virologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Evolução Molecular , Furina/química , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , 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 , Estabilidade Proteica , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
13.
Artigo em Inglês | MEDLINE | ID: mdl-32669265

RESUMO

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Inibidores de Proteases/química , Pirrolidinas/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Chlorocebus aethiops , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Expressão Gênica , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirrolidinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
14.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
15.
BMC Infect Dis ; 20(1): 493, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650739

RESUMO

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. METHODS: 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. RESULTS: In mice vaccinated with hexavalent vaccine, strong total IgG (P < 0.0001) and IgG2a (P < 0.001) responses were induced compared to controls, the ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.001). The survival time of the vaccinated mice increased to 8.38 ± 2.13 days which was significantly higher than controls (P < 0.01). CONCLUSIONS: Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Protozoárias/farmacologia , Toxoplasmose/prevenção & controle , Vacinas de DNA/farmacologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/sangue , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Vacinas de DNA/imunologia
16.
Arch Virol ; 165(9): 2065-2071, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32613291

RESUMO

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterite Viral do Vison/prevenção & controle , Vírus da Enterite do Vison/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/administração & dosagem , Expressão Gênica , Vison/imunologia , Vison/virologia , Enterite Viral do Vison/imunologia , Enterite Viral do Vison/virologia , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência
17.
PLoS One ; 15(6): e0233961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479512

RESUMO

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
18.
Science ; 368(6497): 1386-1392, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32554597

RESUMO

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.


Assuntos
Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 932-941, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567276

RESUMO

Endo-ß-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-ß-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×106 U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.


Assuntos
Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Streptomyces , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética
20.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 959-968, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567279

RESUMO

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.


Assuntos
L-Lactato Desidrogenase , Lactobacillus casei , Ácidos Fenilpirúvicos , Pichia , L-Lactato Desidrogenase/genética , Lactobacillus casei/enzimologia , Lactobacillus casei/genética , Ácidos Fenilpirúvicos/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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