Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.451
Filtrar
1.
Cell Rep ; 32(12): 108185, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32941788

RESUMO

One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays reveal that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of approximately 17,000 SARS-CoV-2 sequences identify a natural variant in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients but also describe the emergence of natural SARS-CoV-2 quasispecies with an extended ORF3b gene that may potentially affect COVID-19 pathogenesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Interferon Tipo I/antagonistas & inibidores , Pneumonia Viral/patologia , Proteínas Virais Reguladoras e Acessórias/genética , Adulto , Sequência de Aminoácidos/genética , Animais , Betacoronavirus/imunologia , Quirópteros/virologia , Códon sem Sentido/genética , Eutérios/virologia , Humanos , Masculino , Pandemias , Proteínas Virais Reguladoras e Acessórias/metabolismo
2.
Vaccine ; 38(41): 6352-6356, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32863070

RESUMO

The world is facing the rising emergency of SARS-CoV-2. The outbreak of COVID-19 has caused a global public health and economic crisis.Recent epidemiological studies have shown that a possible association of BCG vaccination program with decreased COVID-19-related risks, suggesting that BCG may provide protection against COVID-19. Non-specific protection against viral infections is considered as a main mechanism of BCG and clinical trials to determine whether BCG vaccine can protect healthcare workers from the COVID-19 are currently underway. We hypothesized that BCG may carry similar T cell epitopes with SARS-CoV-2 and evaluated the hypothesis by utilizing publicly available database and computer algorithms predicting human leukocyte antigen (HLA) class I-binding peptides. We foundthatBCG contains similar 9-amino acid sequences with SARS-CoV-2. These closely-related peptides had moderate to high binding affinity for multiple common HLA class I molecules, suggesting that cross-reactive T cells against SARS-CoV-2 could be generated by BCG vaccination.


Assuntos
Vacina BCG/imunologia , Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Sequência de Aminoácidos/genética , Betacoronavirus/genética , Reações Cruzadas/imunologia , Epitopos de Linfócito T/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Vacinas Virais/imunologia
3.
PLoS One ; 15(8): e0236477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756607

RESUMO

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.


Assuntos
Anticorpos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos/genética , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos/genética , Humanos , Análise de Célula Única
4.
Int J Mol Sci ; 21(16)2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823591

RESUMO

While SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as the receptor for cell entry, it is important to examine other potential interactions between the virus and other cell receptors. Based on the clinical observation of low prevalence of smoking among hospitalized COVID-19 patients, we examined and identified a "toxin-like" amino acid (aa) sequence in the Receptor Binding Domain of the Spike Glycoprotein of SARS-CoV-2 (aa 375-390), which is homologous to a sequence of the Neurotoxin homolog NL1, one of the many snake venom toxins that are known to interact with nicotinic acetylcholine receptors (nAChRs). We present the 3D structural location of this "toxin-like" sequence on the Spike Glycoprotein and the superposition of the modelled structure of the Neurotoxin homolog NL1 and the SARS-CoV-2 Spike Glycoprotein. We also performed computational molecular modelling and docking experiments using 3D structures of the SARS-CoV-2 Spike Glycoprotein and the extracellular domain of the nAChR α9 subunit. We identified a main interaction between the aa 381-386 of the SARS-CoV-2 Spike Glycoprotein and the aa 189-192 of the extracellular domain of the nAChR α9 subunit, a region which forms the core of the "toxin-binding site" of the nAChRs. The mode of interaction is very similar to the interaction between the α9 nAChR and α-bungarotoxin. A similar interaction was observed between the pentameric α7 AChR chimera and SARS-CoV-2 Spike Glycoprotein. The findings raise the possibility that SARS-CoV-2 may interact with nAChRs, supporting the hypothesis of dysregulation of the nicotinic cholinergic system being implicated in the pathophysiology of COVID-19. Nicotine and other nicotinic cholinergic agonists may protect nAChRs and thus have therapeutic value in COVID-19 patients.


Assuntos
Betacoronavirus/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos/genética , Biologia Computacional , Infecções por Coronavirus/fisiopatologia , Humanos , Simulação de Acoplamento Molecular , Neurotoxinas/genética , Neurotoxinas/metabolismo , Pandemias , Pneumonia Viral/fisiopatologia , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Venenos de Serpentes/genética
5.
PLoS Genet ; 16(8): e1008991, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797042

RESUMO

Accounting for continual evolution of deleterious L1 retrotransposon families, which can contain hundreds to thousands of members remains a major issue in mammalian biology. L1 activity generated upwards of 40% of some mammalian genomes, including humans where they remain active, causing genetic defects and rearrangements. L1 encodes a coiled coil-containing protein that is essential for retrotransposition, and the emergence of novel primate L1 families has been correlated with episodes of extensive amino acid substitutions in the coiled coil. These results were interpreted as an adaptive response to maintain L1 activity, however its mechanism remained unknown. Although an adventitious mutation can inactivate coiled coil function, its effect could be buffered by epistatic interactions within the coiled coil, made more likely if the family contains a diverse set of coiled coil sequences-collectively referred to as the coiled coil sequence space. Amino acid substitutions that do not affect coiled coil function (i.e., its phenotype) could be "hidden" from (not subject to) purifying selection. The accumulation of such substitutions, often referred to as cryptic genetic variation, has been documented in various proteins. Here we report that this phenomenon was in effect during the latest episode of primate coiled coil evolution, which occurred 30-10 MYA during the emergence of primate L1Pa7-L1Pa3 families. First, we experimentally demonstrated that while coiled coil function (measured by retrotransposition) can be eliminated by single epistatic mutations, it nonetheless can also withstand extensive amino acid substitutions. Second, principal component and cluster analysis showed that the coiled coil sequence space of each of the L1Pa7-3 families was notably increased by the presence of distinct, coexisting coiled coil sequences. Thus, sampling related networks of functional sequences rather than traversing discrete adaptive states characterized the persistence L1 activity during this evolutionary event.


Assuntos
Evolução Molecular , Elementos Nucleotídeos Longos e Dispersos/genética , Primatas/genética , Retroelementos/genética , Sequência de Aminoácidos/genética , Animais , Análise Mutacional de DNA , Humanos , Mutação/genética , Proteínas
6.
Sci Rep ; 10(1): 14004, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814791

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel evolutionary divergent RNA virus, is responsible for the present devastating COVID-19 pandemic. To explore the genomic signatures, we comprehensively analyzed 2,492 complete and/or near-complete genome sequences of SARS-CoV-2 strains reported from across the globe to the GISAID database up to 30 March 2020. Genome-wide annotations revealed 1,516 nucleotide-level variations at different positions throughout the entire genome of SARS-CoV-2. Moreover, nucleotide (nt) deletion analysis found twelve deletion sites throughout the genome other than previously reported deletions at coding sequence of the ORF8 (open reading frame), spike, and ORF7a proteins, specifically in polyprotein ORF1ab (n = 9), ORF10 (n = 1), and 3´-UTR (n = 2). Evidence from the systematic gene-level mutational and protein profile analyses revealed a large number of amino acid (aa) substitutions (n = 744), demonstrating the viral proteins heterogeneous. Notably, residues of receptor-binding domain (RBD) showing crucial interactions with angiotensin-converting enzyme 2 (ACE2) and cross-reacting neutralizing antibody were found to be conserved among the analyzed virus strains, except for replacement of lysine with arginine at 378th position of the cryptic epitope of a Shanghai isolate, hCoV-19/Shanghai/SH0007/2020 (EPI_ISL_416320). Furthermore, our results of the preliminary epidemiological data on SARS-CoV-2 infections revealed that frequency of aa mutations were relatively higher in the SARS-CoV-2 genome sequences of Europe (43.07%) followed by Asia (38.09%), and North America (29.64%) while case fatality rates remained higher in the European temperate countries, such as Italy, Spain, Netherlands, France, England and Belgium. Thus, the present method of genome annotation employed at this early pandemic stage could be a promising tool for monitoring and tracking the continuously evolving pandemic situation, the associated genetic variants, and their implications for the development of effective control and prophylaxis strategies.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Heterogeneidade Genética , Genoma Viral/genética , Estudo de Associação Genômica Ampla/métodos , Saúde Global , Pneumonia Viral/epidemiologia , Sequência de Aminoácidos/genética , Anticorpos Neutralizantes/imunologia , Pareamento Incorreto de Bases , Sequência de Bases/genética , Clima , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
PLoS One ; 15(7): e0236366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702062

RESUMO

Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-ß sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-ß sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-ß sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.


Assuntos
Sequência de Aminoácidos/genética , DNA/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Bases , Fragmentação do DNA , DNA Complementar/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
8.
Viruses ; 12(6)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32549200

RESUMO

As more cases of COVID-19 are studied and treated worldwide, it had become apparent that the lethal and most severe cases of pneumonia are due to an out-of-control inflammatory response to the SARS-CoV-2 virus. I explored the putative causes of this specific feature through a detailed genomic comparison with the closest SARS-CoV-2 relatives isolated from bats, as well as previous coronavirus strains responsible for the previous epidemics (SARS-CoV and MERS-CoV). The high variability region of the nsp3 protein was confirmed to exhibit the most variations between closest strains. It was then studied in the context of physiological and molecular data available in the literature. A number of convergent findings suggest de-mono-ADP-ribosylation (de-MARylation) of STAT1 by the SARS-CoV-2 nsp3 as a putative cause of the cytokine storm observed in the most severe cases of COVID-19. This may suggest new therapeutic approaches and help in designing assays to predict the virulence of naturally circulating SARS-like animal coronaviruses.


Assuntos
ADP-Ribosilação/fisiologia , Betacoronavirus/genética , Síndrome da Liberação de Citocina/patologia , Fator de Transcrição STAT1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos/genética , Infecções por Coronavirus/patologia , Humanos , Inflamação/patologia , Inflamação/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Vírus da SARS/genética , Homologia de Sequência , Proteínas não Estruturais Virais/genética
9.
Gene ; 755: 144883, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32565321

RESUMO

The anti-anti-sigma factor BldG has a pleiotropic function in Streptomyces coelicolor A3(2), regulating both morphological and physiological differentiation. Together with the anti-sigma factor UshX, it participates in a partner-switching activation of the sigma factor σH, which has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2). In addition to UshX, BldG also interacts with the anti-sigma factor ApgA, although no target sigma factor has yet been identified. However, neither UshX nor ApgA phosphorylates BldG. This phosphorylation is provided by the anti-sigma factor RsfA, which is specific for the late developmental sigma factor σF. However, BldG is phosphorylated in the rsfA mutant, suggesting that some other anti-sigma factors containing HATPase_c kinase domain are capable to phosphorylate BldG in vivo. Bacterial two-hybrid system (BACTH) was therefore used to investigate the interactions of all suitable anti-sigma factors of S. coelicolor A3(2) with BldG. At least 15 anti-sigma factors were found to interact with BldG. These interactions were confirmed by native PAGE. In addition to RsfA, BldG is specifically phosphorylated on the conserved phosphorylation Ser57 residue by at least seven additional anti-sigma factors. However, only one of them, SCO7328, has been shown to interact with three sigma factors, σG, σK and σM. A mutant with deleted SCO7328 gene was prepared in S. coelicolor A3(2), however, no specific function of SCO7328 in growth, differentiation or stress response could be attributed to this anti-sigma factor. These results suggest that BldG is specifically phosphorylated by several anti-sigma factors and it plays a role in the regulation of several sigma factors in S. coelicolor A3(2). This suggests a complex regulation of the stress response and differentiation in S. coelicolor A3(2) through this pleiotropic anti-sigma factor.


Assuntos
Fator sigma/genética , Streptomyces coelicolor/imunologia , Streptomyces coelicolor/metabolismo , Sequência de Aminoácidos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Regulação Bacteriana da Expressão Gênica/genética , Pleiotropia Genética/genética , Fosforilação/genética , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas/genética , Fator sigma/imunologia , Fator sigma/metabolismo , Streptomyces/genética , Streptomyces coelicolor/genética , Transcrição Genética/genética
10.
Proc Natl Acad Sci U S A ; 117(27): 15731-15739, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561643

RESUMO

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.


Assuntos
Arginina/química , Nucleoproteínas/genética , Ornitina/química , Peptídeos/genética , Sequência de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Arginina/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/química , Ornitina/genética , Peptídeos/química , Proteínas/química , Proteínas/genética , RNA/química , RNA/genética
11.
J Microbiol Immunol Infect ; 53(3): 425-435, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: covidwho-186735

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged in Chinese people in December 2019 and has currently spread worldwide causing the COVID-19 pandemic with more than 150,000 deaths. In order for a SARS-CoV like virus circulating in wild life for a very long time to infect the index case-patient, a number of conditions must be met, foremost among which is the encounter with humans and the presence in homo sapiens of a cellular receptor allowing the virus to bind. Recently it was shown that the SARS-CoV-2 spike protein, binds to the human angiotensin I converting enzyme 2 (ACE2). This molecule is a peptidase expressed at the surface of lung epithelial cells and other tissues, that regulates the renin-angiotensin-aldosterone system. Humans are not equal with respect to the expression levels of the cellular ACE2. Moreover, ACE2 polymorphisms were recently described in human populations. Here we review the most recent evidence that ACE2 expression and/or polymorphism could influence both the susceptibility of people to SARS-CoV-2 infection and the outcome of the COVID-19 disease. Further exploration of the relationship between the virus, the peptidase function of ACE2 and the levels of angiotensin II in SARS-CoV-2 infected patients should help to better understand the pathophysiology of the disease and the multi-organ failures observed in severe COVID-19 cases, particularly heart failure.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Predisposição Genética para Doença/genética , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Sequência de Aminoácidos/genética , Feminino , Insuficiência Cardíaca/virologia , Humanos , Masculino , Insuficiência de Múltiplos Órgãos/virologia , Pandemias , Polimorfismo de Nucleotídeo Único/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral
12.
Mol Cell ; 78(5): 890-902.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32416068

RESUMO

Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations. Our work reveals that strong acidic ADs contain multiple clusters of hydrophobic residues near acidic side chains, explaining why ADs often have a biased amino acid composition. ADs likely use a binding mechanism similar to avidity where a minimum number of weak dynamic interactions are required between activator and target to generate biologically relevant affinity and in vivo function. This mechanism explains the basis for fuzzy binding observed between acidic ADs and targets.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Fatores de Transcrição/genética , Ativação Transcricional/genética , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/metabolismo , Aprendizado Profundo , Ligação Proteica , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia
13.
Gene ; 752: 144788, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32439375

RESUMO

Primulina genus is an ideal wild ornamental flower and emerging model for studying biosynthesis, diversity, and evolution of flower pigment. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation in Primulina remains unknown. Here, changes in anthocyanin content and the expression profiles of anthocyanin biosynthetic structural genes were examined in developing Primulina swinglei flowers and three other organs. Seventy-three R2R3-MYB transcription factor genes were identified from transcriptome of P. swinglei flowers, two of which, PsMYB1 and PsMYB2, are candidate regulators of anthocyanin biosynthesis according to clustering analysis. Furthermore, transient over-expression studies using tobacco leaves showed distinct pigment accumulation following co-infection with PsMYB1 and MrbHLH1 (a previously confirmed anthocyanin regulator from Morella rubra). Additionally, dual luciferase assays showed that PsMYB1 trans-activated the PsANS promoter, with the addition of MrbHLH1 resulting in a 5-fold increase in the intensity of this interaction. PsMYB1 did not, however, have any effect on the PsF3H promoter. The expression profile and dual luciferase assays showed that PsMYB2 plays no roles in anthocyanin regulation. Therefore, PsMYB1 is proposed to be the transcription factor gene regulating anthocyanin biosynthesis in P. swinglei.


Assuntos
Antocianinas/biossíntese , Antocianinas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos/genética , Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Lamiales/genética , Magnoliopsida/genética , Pigmentação/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Transativadores/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética
14.
Gene ; 752: 144791, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32439378

RESUMO

Prkaca consists of the catalytic subunit alpha protein kinase A (PKA), which is involved in many cellular processes. In this study, the cDNA and genomic sequences of prkaca in tilapia hybrids (Oreochromis mossambicus × Oreochromis hornorum) were cloned and analysed. The results showed the prkaca gene consists of 11 exons and 10 introns, and its protein contains 351 amino acid residues and is clustered with Oreochromis niloticus, Maylandia zebra and Haplochromis burtoni first in a phylogenetic tree. Amino acid alignment indicates that prkaca shares the highest identity (100%) to Oreochromis niloticus, Maylandia zebra and Haplochromis burtoni. Two CpG islands of prkaca were found by MethPrimer software, and 32 CG sites were found in the proximal promoter. The methylation level of prkaca in the hybrids (0.31%) was significantly lower than that of their parents (0.94% and 3.43%) in kidney tissue (P < 0.05). The gene expression levels and DNA methylation levels of prkaca in muscle and kidney tissues of the tilapia hybrids were detected by quantitative real-time PCR and bisulfite sequencing PCR and showed a negative correlation under saline-alkali stress. The results of this research demonstrated that DNA methylation levels and prkaca mRNA expression levels were inversely correlated under saline-alkali stress, implying that heterosis is likely accompanied by DNA methylation alterations. This research provides new clues for further investigations of DNA methylation and heterosis in hybrid fish.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Osmorregulação/genética , Tilápia/genética , Sequência de Aminoácidos/genética , Animais , Quimera/genética , China , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metilação de DNA/genética , DNA Complementar/genética , Feminino , Genômica , Vigor Híbrido/genética , Masculino , Filogenia , Regiões Promotoras Genéticas/genética , Equilíbrio Hidroeletrolítico/genética
15.
J Microbiol Immunol Infect ; 53(3): 425-435, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32414646

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged in Chinese people in December 2019 and has currently spread worldwide causing the COVID-19 pandemic with more than 150,000 deaths. In order for a SARS-CoV like virus circulating in wild life for a very long time to infect the index case-patient, a number of conditions must be met, foremost among which is the encounter with humans and the presence in homo sapiens of a cellular receptor allowing the virus to bind. Recently it was shown that the SARS-CoV-2 spike protein, binds to the human angiotensin I converting enzyme 2 (ACE2). This molecule is a peptidase expressed at the surface of lung epithelial cells and other tissues, that regulates the renin-angiotensin-aldosterone system. Humans are not equal with respect to the expression levels of the cellular ACE2. Moreover, ACE2 polymorphisms were recently described in human populations. Here we review the most recent evidence that ACE2 expression and/or polymorphism could influence both the susceptibility of people to SARS-CoV-2 infection and the outcome of the COVID-19 disease. Further exploration of the relationship between the virus, the peptidase function of ACE2 and the levels of angiotensin II in SARS-CoV-2 infected patients should help to better understand the pathophysiology of the disease and the multi-organ failures observed in severe COVID-19 cases, particularly heart failure.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Predisposição Genética para Doença/genética , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Sequência de Aminoácidos/genética , Feminino , Insuficiência Cardíaca/virologia , Humanos , Masculino , Insuficiência de Múltiplos Órgãos/virologia , Pandemias , Polimorfismo de Nucleotídeo Único/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral
16.
PLoS Comput Biol ; 16(5): e1007775, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413045

RESUMO

The human genome harbors a variety of genetic variations. Single-nucleotide changes that alter amino acids in protein-coding regions are one of the major causes of human phenotypic variation and diseases. These single-amino acid variations (SAVs) are routinely found in whole genome and exome sequencing. Evaluating the functional impact of such genomic alterations is crucial for diagnosis of genetic disorders. We developed DeepSAV, a deep-learning convolutional neural network to differentiate disease-causing and benign SAVs based on a variety of protein sequence, structural and functional properties. Our method outperforms most stand-alone programs, and the version incorporating population and gene-level information (DeepSAV+PG) has similar predictive power as some of the best available. We transformed DeepSAV scores of rare SAVs in the human population into a quantity termed "mutation severity measure" for each human protein-coding gene. It reflects a gene's tolerance to deleterious missense mutations and serves as a useful tool to study gene-disease associations. Genes implicated in cancer, autism, and viral interaction are found by this measure as intolerant to mutations, while genes associated with a number of other diseases are scored as tolerant. Among known disease-associated genes, those that are mutation-intolerant are likely to function in development and signal transduction pathways, while those that are mutation-tolerant tend to encode metabolic and mitochondrial proteins.


Assuntos
Doença/genética , Previsões/métodos , Genoma Humano/genética , Alelos , Sequência de Aminoácidos/genética , Biologia Computacional/métodos , Aprendizado Profundo , Redes Reguladoras de Genes/genética , Humanos , Mutação/genética , Mutação de Sentido Incorreto/genética , Rede Nervosa , Fases de Leitura Aberta/genética , Análise de Sequência/métodos , Sequenciamento Completo do Exoma/métodos
17.
PLoS One ; 15(5): e0231813, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442167

RESUMO

The interactions between membrane receptors and their endogenous ligands are key interactions in organisms. Recently, we have shown that a high number of genes encoding human receptors appeared at the same moment as their ligand in the animal tree of life. However, a set of receptors appeared before their present ligand. Different scenarios have been proposed to explain how a receptor can be conserved if its ligand is not yet appeared. However, these scenarios have been proposed individually and have never been studied in a global way. In this study, we investigated 30 mammalian pairs of ligand/receptor for which the first ligand appeared after its receptor in the tree of life, by using common indexes of selection, and proposed different scenarios explaining the earlier appearance of a receptor relative to its ligand. Based on 3D structural studies, our indexes allowed us to classify the evolution of these partners into different scenarios: 1) a scenario where the binding interface of the receptor is already present and under purifying selection before the appearance of the ligand; 2) a scenario where the binding interface seems to have appeared progressively, and 3) a scenario where the binding site seems to have been reshuffled since its appearance. As some scenarios were confirmed by the literature, we concluded that simple indexes can give a good highlight of the evolutive history of two partners that did not appear at the same time. Based on these scenarios, we also hypothesize that the replacement of a ligand by another is a frequent phenomenon during evolution.


Assuntos
Sítios de Ligação/genética , Evolução Molecular , Ligantes , Ligação Proteica/genética , Receptores de Superfície Celular , Sequência de Aminoácidos/genética , Animais , Simulação por Computador , Humanos , Modelos Moleculares , Conformação Molecular , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
18.
BMC Med Genet ; 21(1): 95, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32380971

RESUMO

BACKGROUND: Conotruncal heart defects (CTDs) are a group of congenital heart malformations that cause anomalies of cardiac outflow tracts. In the past few decades, many genes related to CTDs have been reported. Serum response factor (SRF) is a ubiquitous nuclear protein that acts as transcription factor, and SRF was found to be a critical factor in heart development and to be strongly expressed in the myocardium of the developing mouse and chicken hearts. The targeted inactivation of SRF during heart development leads to embryonic lethality and myocardial defects in mice. METHODS: To illustrate the relationship between SRF and human heart defects, we screened SRF mutations in 527 CTD patients, a cross sectional study. DNA was extracted from peripheral leukocyte cells for target sequencing. The mutations of SRF were detected and validated by Sanger sequencing. The affection of the mutations on wild-type protein was analyzed by in silico softwares. Western blot and real time PCR were used to analyze the changes of the expression of the mutant mRNA and protein. In addition, we carried out dual luciferase reporter assay to explore the transcriptional activity of the mutant SRF. RESULTS: Among the target sequencing results of 527 patients, two novel mutations (Mut1: c.821A > G p.G274D, the adenine(A) was mutated to guanine(G) at position 821 of the SRF gene coding sequences (CDS), lead to the Glycine(G) mutated to Asparticacid(D) at position 274 of the SRF protein amino acid sequences; Mut2: c.880G > T p.G294C, the guanine(G) was mutated to thymine (T) at position 880 of the SRF CDS, lead to the Glycine(G) mutated to Cysteine (C) at position 294 of the SRF protein amino acid sequences.) of SRF (NM_003131.4) were identified. Western blotting and real-time PCR showed that there were no obvious differences between the protein expression and mRNA transcription of mutants and wild-type SRF. A dual luciferase reporter assay showed that both SRF mutants (G274D and G294C) impaired SRF transcriptional activity at the SRF promoter and atrial natriuretic factor (ANF) promoter (p < 0.05), additionally, the mutants displayed reduced synergism with GATA4. CONCLUSION: These results suggest that SRF-p.G274D and SRF-p.G294C may have potential pathogenic effects.


Assuntos
Fator Natriurético Atrial/genética , Fator de Transcrição GATA4/genética , Cardiopatias Congênitas/genética , Fator de Resposta Sérica/genética , Sequência de Aminoácidos/genética , Animais , Estudos Transversais , Feminino , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Cardiopatias Congênitas/patologia , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Miocárdio/metabolismo , Miocárdio/patologia , Regiões Promotoras Genéticas/genética
19.
J Med Microbiol ; 69(7): 986-998, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32459617

RESUMO

Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines.Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness.Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented.Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six-ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four-nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences.Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.


Assuntos
Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Influenza Humana/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Antígenos Virais/genética , Bulgária/epidemiologia , Monitoramento Epidemiológico , Evolução Molecular , Variação Genética/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , História do Século XXI , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/imunologia , Influenza Humana/história , Influenza Humana/virologia , Neuraminidase/genética , Filogenia , RNA Viral/genética , Estações do Ano , Análise de Sequência de DNA/métodos
20.
Nat Commun ; 11(1): 1725, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265442

RESUMO

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.


Assuntos
Glutarredoxinas/química , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos/genética , Catálise , Domínio Catalítico/genética , Dissulfetos/química , Ativação Enzimática , Ensaios Enzimáticos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/química , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica em alfa-Hélice , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA