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1.
Med Sci (Paris) ; 36(8-9): 783-796, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32773024

RESUMO

SARS-CoV-2 is a new human coronavirus (CoV), which emerged in People's Republic of China at the end of 2019 and is responsible for the global Covid-19 pandemic that caused more than 540 000 deaths in six months. Understanding the origin of this virus is an important issue and it is necessary to determine the mechanisms of its dissemination in order to be able to contain new epidemics. Based on phylogenetic inferences, sequence analysis and structure-function relationships of coronavirus proteins, informed by the knowledge currently available, we discuss the different scenarios evoked to account for the origin - natural or synthetic - of the virus. On the basis of currently available data, it is impossible to determine whether SARS-CoV-2 is the result of a natural zoonotic emergence or an accidental escape from experimental strains. Regardless of its origin, the study of the evolution of the molecular mechanisms involved in the emergence of this pandemic virus is essential to develop therapeutic and vaccine strategies.


Assuntos
Betacoronavirus/genética , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/virologia , Coronavirus/classificação , Evolução Molecular , Pandemias , Filogenia , Pneumonia Viral/virologia , RNA Viral/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , Derramamento de Material Biológico , China/epidemiologia , Infecções por Coronaviridae/transmissão , Infecções por Coronaviridae/veterinária , Infecções por Coronaviridae/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Reservatórios de Doenças , Mutação com Ganho de Função , Genoma Viral , HIV/genética , Especificidade de Hospedeiro , Humanos , Mamíferos/virologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Vírus Reordenados/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Zoonoses
2.
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
3.
Med Hypotheses ; 143: 110151, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763661

RESUMO

Today it remains unclear why children seem to be less likely to get infected by COVID-19 or why they appear to be less symptomatic after infections. All individuals, especially children, are exposed to various viruses including human coronavirus (CoVs) that can generally lead to respiratory infections. We hypothesize that recurrent CoVs exposure may induce an effective antiviral B and T-cell-mediated adaptive immune response, which could also be protective against COVID-19. Based on the high-homology between the Spike protein epitopes of taxonomically-related coronaviruses, we theorize that past/recurrent contact with CoVs might shield children also against the circulating COVID-19 through a possible neutralizing antibody response previously CoVs-induced. This would open up possible lines of research for the development of live-attenuated virus vaccines from CoVs. Future research is desirable to confirm or disprove such hypothesis.


Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Memória Imunológica , Modelos Imunológicos , Pandemias , Pneumonia Viral/epidemiologia , Adulto , Distribuição por Idade , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Betacoronavirus/genética , Betacoronavirus/imunologia , Linfócitos T CD4-Positivos/imunologia , Criança , Coronavirus/genética , Coronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Reações Cruzadas , Resistência à Doença , Epitopos/genética , Epitopos/imunologia , Humanos , Peptidil Dipeptidase A/análise , Pneumonia Viral/imunologia , Alvéolos Pulmonares/química , Receptores Virais/análise , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Atenuadas , Vacinas Virais
4.
Biochem Biophys Res Commun ; 532(1): 134-138, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32829876

RESUMO

SARS-CoV-2 is the etiologic agent of COVID-19. There is currently no effective means of preventing infections by SARS-CoV-2, except through restriction of population movement and contact. An understanding of the origin, evolution and biochemistry (molecular biology) of SARS-CoV-2 is a prerequisite to its control. Mutations in the phosphorylation sites of SARS-CoV-2 encoded nucleocapsid protein isolated from various populations and locations, are described. Mutations occurred in the phosphorylation sites, all located within a stretch which forms a phosphorylation dependent interaction site, including C-TAK1 phosphorylation sites for 14-3-3. The consequences of these mutations are discussed and a structure-based model for the role of protein 14-3-3 in the sequestration and inhibition of SARS-CoV-2 nucleocapsid protein's function is presented. It is proposed that the phosphorylation of SARS-CoV-2 nucleocapsid protein and its sequestration by Protein 14-3-3 is a cellular response mechanism for the control and inhibition of the replication, transcription and packaging of the SARS-CoV-2 genome.


Assuntos
Proteínas 14-3-3/química , Betacoronavirus/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Proteínas do Nucleocapsídeo/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosforilação , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
5.
PLoS One ; 15(8): e0237744, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841246

RESUMO

Both the Mediterranean (MED) species of the Bemisia tabaci whitefly complex and the greenhouse whitefly (Trialeurodes vaporariorum, TV) are important agricultural pests. The two species of whiteflies differ in many aspects such as morphology, geographical distribution, host plant range, plant virus transmission, and resistance to insecticides. However, the molecular basis underlying their differences remains largely unknown. In this study, we analyzed the genetic divergences between the transcriptomes of MED and TV. In total, 2,944 pairs of orthologous genes were identified. The average identity of amino acid sequences between the two species is 93.6%. The average nonsynonymous (Ka) and synonymous (Ks) substitution rates and the ratio of Ka/Ks of the orthologous genes are 0.0389, 2.23 and 0.0204, respectively. The low average Ka/Ks ratio indicates that orthologous genes tend to be under strong purified selection. The most divergent gene classes are related to the metabolisms of xenobiotics, cofactors, vitamins and amino acids, and this divergence may underlie the different biological characteristics between the two species of whiteflies. Genes of differential expression between the two species are enriched in carbohydrate metabolism and regulation of autophagy. These findings provide molecular clues to uncover the biological and molecular differences between the two species of whiteflies.


Assuntos
Produção Agrícola , Genes de Insetos/genética , Especiação Genética , Hemípteros/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Região do Mediterrâneo , Anotação de Sequência Molecular , RNA-Seq , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Vitaminas/metabolismo , Xenobióticos/metabolismo
6.
Infect Genet Evol ; 84: 104493, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32768565

RESUMO

The emergence of COVID-19 has triggered many works aiming at identifying the animal intermediate potentially involved in the transmission of SARS-CoV-2 to humans. The presence of SARS-CoV-2-related viruses in Malayan pangolins, in silico analysis of the ACE2 receptor polymorphism and sequence similarities between the Receptor Binding Domain (RBD) of the spike proteins of pangolin and human Sarbecoviruses led to the proposal of pangolin as intermediary. However, the binding affinity of the pangolin ACE2 receptor for SARS-CoV-2 RBD was later on reported to be low. Here, we provide evidence that the pangolin is not the intermediate animal at the origin of the human pandemic. Moreover, data available do not fit with the spillover model currently proposed for zoonotic emergence which is thus unlikely to account for this outbreak. We propose a different model to explain how SARS-CoV-2 related coronaviruses could have circulated in different species, including humans, before the emergence of COVID-19.


Assuntos
Betacoronavirus/genética , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Eutérios/virologia , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/epidemiologia , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/classificação , Betacoronavirus/metabolismo , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/metabolismo , Zoonoses
7.
Biophys Chem ; 264: 106425, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663708

RESUMO

The novel SARS-CoV-2 is the etiological agent causing the Coronavirus disease 2019 (COVID-19), which continues to become an inevitable pandemic outbreak. Over a short span of time, the structures of therapeutic target proteins for SARS-CoV-2 were identified based on the homology modelled structure of similar SARS-CoV transmission of 2003. Since the onset of the disease, the research community has been looking for a potential drug lead. Out of all the known resolved structures related to SARS-CoV, Main protease (Mpro) is considered an attractive anti-viral drug target on the grounds of its role in viral replication and probable non-interactive competency to bind to any viral host protein. To the best of our knowledge, till date only one compound has been identified and tested in-vivo as a potent inhibitor of Mpro protein, addressed as N3 (PubChem Compound CID: 6323191) and is known to bind irreversibly to Mpro suppressing its activity. Using computational approach, we intend to identify a probable natural fungal metabolite to interact and inhibit Mpro. After screening various small molecules for molecular docking and dynamics simulation, we propose Pyranonigrin A, a secondary fungal metabolite to possess potent inhibitory potential against the Main protease (Mpro) expressed in SARS-CoV-2 virus.


Assuntos
Antivirais/química , Betacoronavirus/enzimologia , Inibidores de Proteases/química , Pironas/química , Pirróis/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Betacoronavirus/patogenicidade , Sítios de Ligação , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Expressão Gênica , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
8.
Nat Commun ; 11(1): 3317, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620775

RESUMO

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.


Assuntos
Embrião não Mamífero/metabolismo , Mitose , Receptores de Peptídeos/metabolismo , Esfingolipídeos/metabolismo , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Divisão Celular Assimétrica/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Lipoilação , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismo , Receptores de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Fuso Acromático/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Nat Commun ; 11(1): 3301, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620849

RESUMO

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação/genética , Células Cultivadas , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética
10.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
11.
BMC Evol Biol ; 20(1): 85, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664916

RESUMO

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transportation of various endogenous or exogenous substances. Two ABCG2 gene subfamily members have been identified in birds. A detailed comparative study of the ABCG2 and ABCG2-like genes aid our understanding of their evolutionary history at the molecular level and provide a theoretical reference for studying the specific functions of ABCG2 and ABCG2-like genes in birds. RESULTS: We first identified 77 ABCG2/ABCG2-like gene sequences in the genomes of 41 birds. Further analysis showed that both the nucleic acid and amino acid sequences of ABCG2 and ABCG2-like genes were highly conserved and exhibited high homology in birds. However, significant differences in the N-terminal structure were found between the ABCG2 and ABCG2-like amino acid sequences. A selective pressure analysis showed that the ABCG2 and ABCG2-like genes were affected by purifying selection during the process of bird evolution. CONCLUSIONS: We believe that multiple members of the ABCG2 gene subfamily exist on chromosome 4 in the ancestors of birds. Over the long course of evolution, only the ABCG2 gene was retained on chromosome 4 in birds. The ABCG2-like gene on chromosome 6 might have originated from chromosome replication or fusion. The structural differences between the N terminus of ABCG2 protein and those of ABCG2-like proteins might lead to functional differences between the corresponding genes.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Aves/genética , Evolução Molecular , Homologia de Sequência de Aminoácidos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Cromossomos/genética , Sequência Conservada/genética , Éxons/genética , Regulação da Expressão Gênica , Genoma , Íntrons/genética , Família Multigênica , Fases de Leitura Aberta/genética , Fosforilação , Filogenia , Domínios Proteicos , Sítios de Splice de RNA/genética , Seleção Genética , Sintenia/genética
12.
PLoS One ; 15(6): e0235153, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603341

RESUMO

The secondary structure prediction of proteins is a classic topic of computational structural biology with a variety of applications. During the past decade, the accuracy of prediction achieved by state-of-the-art algorithms has been >80%; meanwhile, the time cost of prediction increased rapidly because of the exponential growth of fundamental protein sequence data. Based on literature studies and preliminary observations on the relationships between the size/homology of the fundamental protein dataset and the speed/accuracy of predictions, we raised two hypotheses that might be helpful to determine the main influence factors of the efficiency of secondary structure prediction. Experimental results of size and homology reductions of the fundamental protein dataset supported those hypotheses. They revealed that shrinking the size of the dataset could substantially cut down the time cost of prediction with a slight decrease of accuracy, which could be increased on the contrary by homology reduction of the dataset. Moreover, the Shannon information entropy could be applied to explain how accuracy was influenced by the size and homology of the dataset. Based on these findings, we proposed that a proper combination of size and homology reductions of the protein dataset could speed up the secondary structure prediction while preserving the high accuracy of state-of-the-art algorithms. Testing the proposed strategy with the fundamental protein dataset of the year 2018 provided by the Universal Protein Resource, the speed of prediction was enhanced over 20 folds while all accuracy measures remained equivalently high. These findings are supposed helpful for improving the efficiency of researches and applications depending on the secondary structure prediction of proteins. To make future implementations of the proposed strategy easy, we have established a database of size and homology reduced protein datasets at http://10.life.nctu.edu.tw/UniRefNR.


Assuntos
Estrutura Secundária de Proteína , Proteínas/química , Algoritmos , Biologia Computacional , Confiabilidade dos Dados , Bases de Dados de Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
Nat Commun ; 11(1): 3328, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620864

RESUMO

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.


Assuntos
Variação Genética , Proteínas de Membrana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Isoformas de RNA/genética , Retina/metabolismo , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de RNA/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Degeneração Retiniana/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
15.
Nat Commun ; 11(1): 3784, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728052

RESUMO

The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.


Assuntos
Bacteriófagos/genética , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Edição de Genes/métodos , Interações Hospedeiro-Parasita/genética , Homologia de Sequência de Aminoácidos
16.
Arch Virol ; 165(10): 2229-2239, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32676682

RESUMO

A reexamination of proteins with conserved cysteines and basic amino acids encoded by the 3'-proximal gene of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses has been carried out. The cysteines are involved in a putative Zn-finger domain, which, together with the basic amino acids, form part of the nuclear or nucleolar localization signals. An in-depth study of one of these proteins, p15 from grapevine B virus (GVB), has shown: (i) a three-dimensional structure with four α-helices predicted by two independent in silico approaches, (ii) the nucleolus as the main accumulation site by applying confocal laser microscopy to a fusion between p15 and the green fluorescent protein, (iii) the involvement of the basic amino acids and the putative Zn-finger domain, mapping at the N-terminal region of p15, in the nucleolar localization signal, as revealed by the effect of six alanine substitution mutations, (iv) the p15 suppressor function of sense-mediated RNA silencing as revealed by agroinfiltration in a transgenic line of Nicotiana benthamiana, and (v) the enhancer activity of p15 on viral pathogenicity in N. benthamiana when expressed from a potato virus X vector. In addition, we elaborate on an evolutionary scenario for these filamentous viruses, invoking takeover by a common ancestor(s) of viral or host genes coding for those cysteine-rich proteins, followed by divergence, which would also explain why they are encoded in the 3'-proximal gene of the genomic single-stranded viral RNA.


Assuntos
Flexiviridae/genética , Fases de Leitura Aberta , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Clonagem Molecular , Evolução Molecular , Flexiviridae/metabolismo , Expressão Gênica , Modelos Moleculares , Filogenia , Células Vegetais/virologia , Folhas de Planta/virologia , Conformação Proteica em alfa-Hélice , Domínios Proteicos , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tabaco/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo
17.
Arch Virol ; 165(10): 2259-2277, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32699981

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.


Assuntos
Citocinas/genética , Genoma Viral , Filogenia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Citocinas/imunologia , Evolução Molecular , Expressão Gênica , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Fases de Leitura Aberta , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/patologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Recombinação Genética , República da Coreia/epidemiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Virulência
18.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
19.
Infect Genet Evol ; 84: 104451, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640381

RESUMO

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Proteínas do Nucleocapsídeo/química , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Vírion/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Doxiciclina/química , Doxiciclina/farmacologia , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinoxalinas/química , Quinoxalinas/farmacologia , Rutina/química , Rutina/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Simeprevir/química , Simeprevir/farmacologia , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Vírion/genética , Vírion/metabolismo
20.
PLoS One ; 15(7): e0236201, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687545

RESUMO

Interaction of phospholipase D2 (PLD2) with phosphatidylinositol (4,5)-bisphosphate (PIP2) is regarded as the critical step of numerous physiological processes. Here we build a full-length model of human PLD2 (hPLD2) combining template-based and ab initio modeling techniques and use microsecond all-atom molecular dynamics (MD) simulations of the protein in contact with a complex membrane to determine hPLD2-PIP2 interactions. MD simulations reveal that the intermolecular interactions preferentially occur between specific PIP2 phosphate groups and hPLD2 residues; the most strongly interacting residues are arginine at the pbox consensus sequence (PX) and pleckstrin homology (PH) domain. Interaction networks indicate formation of clusters at the protein-membrane interface consisting of amino acids, PIP2, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA); the largest cluster was in the PH domain.


Assuntos
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/química , Sequência Consenso , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase D/química , Fosfolipase D/ultraestrutura , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos
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