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1.
J Theor Biol ; 505: 110425, 2020 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-32735992

RESUMEN

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.


Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Diseño de Fármacos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Betacoronavirus/genética , Sitios de Unión , Evolución Molecular Dirigida , Humanos , Pandemias , Peptidil-Dipeptidasa A/uso terapéutico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estabilidad Proteica , Glicoproteína de la Espiga del Coronavirus/metabolismo
2.
ACS Nano ; 14(8): 10616-10623, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32806067

RESUMEN

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.


Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/virología , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sustitución de Aminoácidos , Antivirales/química , Antivirales/farmacología , Betacoronavirus/química , Betacoronavirus/genética , Sitios de Unión/genética , Diseño de Fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Humanos , Simulación de Dinámica Molecular , Mutación , Oligopéptidos/química , Oligopéptidos/farmacología , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/fisiología , Dominios Proteicos , Receptores Virales/química , Receptores Virales/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus
3.
J Transl Med ; 18(1): 321, 2020 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-32831104

RESUMEN

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Asunto(s)
Betacoronavirus/fisiología , Mutación Missense , Peptidil-Dipeptidasa A/genética , Dominios y Motivos de Interacción de Proteínas/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sustitución de Aminoácidos , Betacoronavirus/metabolismo , Sitios de Unión/genética , Infecciones por Coronavirus/etnología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Análisis Mutacional de ADN/métodos , Bases de Datos Genéticas , Predisposición Genética a la Enfermedad/etnología , Variación Genética , Geografía , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/etnología , Neumonía Viral/genética , Neumonía Viral/virología , Polimorfismo de Nucleótido Simple , Unión Proteica , Estructura Secundaria de Proteína/genética , Glicoproteína de la Espiga del Coronavirus/química , Internalización del Virus
4.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-32848127

RESUMEN

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.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación , Sustitución de Aminoácidos , Sitios de Unión/genética , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios Proteicos , Pliegue de Proteína , Modificación Traduccional de las Proteínas/genética , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismo , Supresión Genética , Temperatura
5.
PLoS One ; 15(7): e0234792, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32614850

RESUMEN

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.


Asunto(s)
Proteínas Angiogénicas/biosíntesis , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Encéfalo/citología , Proteínas Portadoras/análisis , Linaje de la Célula , Epítopos/inmunología , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Desarrollo de Músculos , Proteína MioD/análisis , Especificidad de Órganos , Conformación Proteica , Dominios Proteicos , Conejos , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Secuencias Repetitivas de Aminoácido , Piel/citología , Especificidad de la Especie , Tatuaje , Adulto Joven
6.
PLoS Genet ; 16(7): e1008901, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32645003

RESUMEN

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.


Asunto(s)
Enfermedades Cerebelosas/genética , Proteínas del Citoesqueleto/genética , Drosophila melanogaster/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , Sustitución de Aminoácidos/genética , Animales , Sistemas CRISPR-Cas/genética , Enfermedades Cerebelosas/patología , Cerebelo/metabolismo , Cerebelo/patología , Modelos Animales de Enfermedad , Exosomas/genética , Humanos , Mutación/genética , Neuronas/patología , ARN/genética
7.
BMC Infect Dis ; 20(1): 513, 2020 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-32677899

RESUMEN

BACKGROUND: Imported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China. METHOD: Fifty-five imported falciparum malaria cases in Guizhou Province during 2012-2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software. RESULTS: The prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%. CONCLUSIONS: The high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.


Asunto(s)
Enfermedades Transmisibles Importadas/parasitología , Resistencia a Medicamentos/genética , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas Protozoarias/genética , Adulto , África/epidemiología , Sustitución de Aminoácidos/genética , Antimaláricos/uso terapéutico , China/epidemiología , Estudios de Cohortes , Femenino , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/epidemiología , Masculino , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación , Plasmodium falciparum/aislamiento & purificación , Enfermedad Relacionada con los Viajes
8.
BMC Infect Dis ; 20(1): 518, 2020 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-32677920

RESUMEN

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.


Asunto(s)
Infecciones por Campylobacter/diagnóstico , Campylobacter jejuni/genética , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/uso terapéutico , Mutación Puntual , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sustitución de Aminoácidos , Infecciones por Campylobacter/tratamiento farmacológico , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/aislamiento & purificación , Niño , Ciprofloxacino/uso terapéutico , Análisis Mutacional de ADN/métodos , Diarrea/diagnóstico , Diarrea/tratamiento farmacológico , Diarrea/microbiología , Humanos , Isoleucina/genética , Pruebas de Sensibilidad Microbiana , Perú , Treonina/genética
9.
J Virol ; 94(18)2020 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-32641486

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to enabling implementation of effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage A/G, containing mostly sequences from North America, and lineage B/S, containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized 14 independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represented local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described for the country. Within this local transmission cluster, we also identified an H49Y amino acid change in the Spike protein. This mutation represents a homoplasy occurring independently through time and space and may function as a molecular marker to follow any further spread of these viral variants throughout the country. Our results provide a general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts.IMPORTANCE Understanding the introduction, spread, and establishment of SARS-CoV-2 within distinct human populations as well as the evolution of the pandemics is crucial to implement effective control strategies. In this work, we report that the initial virus strains introduced in Mexico came from Europe and the United States and that the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains with a H49Y mutation in the Spike protein, which could be further used as a molecular marker to follow viral spread within the country and the region.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Variación Genética , Genoma Viral , Genómica , Neumonía Viral/epidemiología , Neumonía Viral/virología , Sustitución de Aminoácidos , Betacoronavirus/clasificación , Biología Computacional/métodos , Infecciones por Coronavirus/transmisión , Genómica/métodos , Humanos , México/epidemiología , Mutación , Pandemias , Filogenia , Neumonía Viral/transmisión
10.
BMC Bioinformatics ; 21(1): 311, 2020 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-32677889

RESUMEN

BACKGROUND: Polyploid organisms such as wheat complicate even the simplest of procedures in molecular biology. Whilst knowledge of genomic sequences in crops is increasing rapidly, the scientific community is still a long way from producing a full pan-genome for every species. Polymerase chain reaction and Sanger sequencing therefore remain widely used as methods for characterizing gene sequences in many varieties of crops. High sequence similarity between genomes in polyploids means that if primers are not homeologue-specific via the incorporation of a SNP at the 3' tail, sequences other than the target sequence will also be amplified. Current consensus for gene cloning in wheat is to manually perform many steps in a long bioinformatics pipeline. RESULTS: Here we present AutoCloner ( www.autocloner.com ), a fully automated pipeline for crop gene cloning that includes a free-to-use web interface for users. AutoCloner takes a sequence of interest from the user and performs a basic local alignment search tool (BLAST) search against the genome assembly for their particular polyploid crop. Homologous sequences are then compiled with the input sequence into a multiple sequence alignment which is mined for single-nucleotide polymorphisms (SNPs). Various combinations of potential primers that cover the entire gene of interest are then created and evaluated by Primer3; the set of primers with the highest score, as well as all possible primers at every SNP location, are then returned to the user for polymerase chain reaction (PCR). We have successfully used AutoCloner to clone various genes of interest in the Apogee wheat variety, which has no current genome sequence. In addition, we have successfully run the pipeline on ~ 80,000 high-confidence gene models from a wheat genome assembly. CONCLUSION: AutoCloner is the first tool to fully-automate primer design for gene cloning in polyploids, where previously the consensus within the wheat community was to perform this process manually. The web interface for AutoCloner provides a simple and effective polyploid primer-design method for gene cloning, with no need for researchers to download software or input any other details other than their sequence of interest.


Asunto(s)
Clonación Molecular , Biología Computacional/métodos , Cartilla de ADN/metabolismo , Poliploidía , Homología de Secuencia , Programas Informáticos , Triticum/genética , Sustitución de Aminoácidos/genética , Secuencia de Bases , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/genética
11.
Cell Rep ; 31(11): 107774, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: covidwho-436427

RESUMEN

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Asunto(s)
Betacoronavirus/enzimología , Microscopía por Crioelectrón , ARN Replicasa/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Escherichia coli/genética , Evolución Molecular , Modelos Moleculares , Complejos Multiproteicos/química , ARN Replicasa/metabolismo , Virus del SRAS/enzimología , Proteínas no Estructurales Virales/metabolismo
12.
Cell Rep ; 31(11): 107774, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: covidwho-594914

RESUMEN

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Asunto(s)
Betacoronavirus/enzimología , Microscopía por Crioelectrón , ARN Replicasa/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Escherichia coli/genética , Evolución Molecular , Modelos Moleculares , Complejos Multiproteicos/química , ARN Replicasa/metabolismo , Virus del SRAS/enzimología , Proteínas no Estructurales Virales/metabolismo
13.
PLoS One ; 15(6): e0235394, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32598352

RESUMEN

Several Amaranthus spp. around the world have evolved resistance (and cross resistance) to various herbicide mechanisms of action. Populations of redroot pigweed (RRPW-R) and tall waterhemp (TW-R) in Mississippi, USA have been suspected to be resistant to one or more acetolactate synthase (ALS) inhibiting herbicides. Whole plant dose-response experiments with multiple ALS inhibitors, ALS enzyme assays with pyrithiobac, and molecular sequence analysis of ALS gene constructs were conducted to confirm and characterize the resistance profile and nature of the mechanism in the RRPW-R and TW-R populations. Two susceptible populations, RRPW-S and TW-S were included for comparison with RRPW-R and TW-R, correspondingly. The resistance index (R/S; the herbicide dose required to reduce plant growth by 50% of resistant population compared to the respective susceptible population) values of the RRPW-R population were 1476, 3500, and 900 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively. The R/S values of the TW-R population for pyrithiobac, imazaquin, and trifloxysulfuron were 51, 950, and 2600, respectively. I50 values of RRPW-S and RRPW-R populations for pyrithiobac were 0.062 and 208.33 µM, indicating that the ALS enzyme of the RRPW-R population is 3360-fold more resistant to pyrithiobac than the RRPW-S population under our experimental conditions. The ALS enzyme of the TW-R population was 1214-fold resistant to pyrithiobac compared to the TW-S population, with the I50 values for pyrithiobac of ALS from TW-R and TW-S populations being 87.4 and 0.072 µM, correspondingly. Sequencing of the ALS gene identified a point mutation at position 574 of the ALS gene leading to substitution of tryptophan (W) residue with a leucine (L) residue in both RRPW-R and TW-R populations. Thus, the RRPW-R and TW-R populations are resistant to several ALS-inhibiting herbicides belonging to different chemical classes due to an altered target site, i.e., ALS. Resistance in Amaranthus spp. to commonly used ALS-inhibiting herbicides warrants an integrated weed management scheme incorporating chemical, mechanical, and cultural strategies by growers.


Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Acetolactato Sintasa/genética , Amaranthus/efectos de los fármacos , Resistencia a los Herbicidas , Herbicidas/farmacología , Mutación , Proteínas de Plantas/antagonistas & inhibidores , Acetolactato Sintasa/metabolismo , Amaranthus/clasificación , Amaranthus/enzimología , Amaranthus/genética , Sustitución de Aminoácidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
14.
PLoS One ; 15(6): e0235133, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32584877

RESUMEN

We have used a combination of computational and structure-based redesign of the low molecular weight protein tyrosine phosphatase, LMW-PTP, to create new activity towards phosphoinositide substrates for which the wild-type enzyme had little or no activity. The redesigned enzymes retain catalytic activity despite residue alterations in the active site, and kinetic experiments confirmed specificity for up to four phosphoinositide substrates. Changes in the shape and overall volume of the active site where critical to facilitate access of the new substrates for catalysis. The kinetics data suggest that both the position and the combination of amino acid mutations are important for specificity towards the phosphoinositide substrates. The introduction of basic residues proved essential to establish new interactions with the multiple phosphate groups in the inositol head, thus promoting catalytically productive complexes. The crystallographic structures of the top-ranking designs confirmed the computational predictions and showed that residue substitutions do not alter the overall folding of the phosphatase or the conformation of the active site P-loop. The engineered LMW-PTP mutants with new activities can be useful reagents in investigating cell signalling pathways and offer the potential for therapeutic applications.


Asunto(s)
Sustitución de Aminoácidos , Simulación por Computador , Fosfatidilinositoles/química , Pliegue de Proteína , Proteínas Tirosina Fosfatasas , Dominio Catalítico , Estructura Secundaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato
15.
PLoS Pathog ; 16(6): e1008592, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32555740

RESUMEN

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Virus de la Influenza B/metabolismo , Infecciones por Orthomyxoviridae , Oseltamivir/farmacología , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Hurones , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Virus de la Influenza B/genética , Macaca fascicularis , Macrólidos , Masculino , Mutación Missense , Neuraminidasa/genética , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología
16.
Cell Rep ; 31(11): 107774, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32531208

RESUMEN

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Asunto(s)
Betacoronavirus/enzimología , Microscopía por Crioelectrón , ARN Replicasa/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Escherichia coli/genética , Evolución Molecular , Modelos Moleculares , Complejos Multiproteicos/química , ARN Replicasa/metabolismo , Virus del SRAS/enzimología , Proteínas no Estructurales Virales/metabolismo
17.
Ann Hematol ; 99(8): 1763-1769, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32577844

RESUMEN

We aimed to detect the MYD88L265P and CXCR4S338X mutations in cell-free DNA (cfDNA) in patients with Waldenström macroglobulinemia (WM). We collected peripheral blood and paired bone marrow aspirates from 27 WM patients (including 16 patients with newly diagnosed WM, 3 patients with WM in relapse and 8 patients with WM during treatment). cfDNA was extracted from peripheral blood using a QIAamp Circulating Nucleic Acid Kit. The MYD88L265P and CXCR4S338X mutations were detected by real-time allele-specific PCR (AS-PCR) in cfDNA and genomic DNA (gDNA) extracted from bone marrow aspirates. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was determined using a serial dilution of 10%, 2%, 0.4% and 0.08% MYD88L265P cfDNA in wild-type cfDNA. Among the 27 patients, MYD88L265P was detected in 88.9% of them in gDNA and in 85.2% of them in cfDNA, with a concordance rate of 96.3%. The concordance rates were 93.8%, 100% and 100% in patients with newly diagnosed WM, patients with WM in relapse and patients with WM during treatment, respectively. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was 0.4%. CXCR4S338X was detected in 6.3% of the 16 newly diagnosed WM patients in both gDNA and cfDNA, with a concordance rate of 100.0%. It is feasible to apply cfDNA to detect MYD88L265P and CXCR4S338X in WM patients with a high concordance rate.


Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Mutación Missense , Factor 88 de Diferenciación Mieloide/genética , Proteínas de Neoplasias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/genética , Macroglobulinemia de Waldenström/genética , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Ácidos Nucleicos Libres de Células/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor 88 de Diferenciación Mieloide/sangre , Proteínas de Neoplasias/sangre , Receptores CXCR4/sangre , Sensibilidad y Especificidad , Macroglobulinemia de Waldenström/sangre , Macroglobulinemia de Waldenström/terapia
18.
PLoS One ; 15(6): e0234192, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32479562

RESUMEN

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.


Asunto(s)
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supresión Genética , Ubiquinona/biosíntesis , Sustitución de Aminoácidos , Asparagina , Medios de Cultivo/química , Regulación Fúngica de la Expresión Génica , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/genética
19.
Biosci Rep ; 40(5)2020 05 29.
Artículo en Inglés | MEDLINE | ID: covidwho-378398

RESUMEN

Since 2002, ß coronaviruses (CoVs) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019). Spike (S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the nCoV2019 takes its course throughout the world, more and more sequence analyses are being done and genome sequences are being deposited in various databases. From India, two clinical isolates have been sequenced and the full genome has been deposited in GenBank. We have performed sequence analyses of the Spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates, one was found to harbor a mutation in its receptor-binding domain (RBD) at position 407. At this site, arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-ß branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor binding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Mutación Puntual , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Betacoronavirus/aislamiento & purificación , Humanos , India , Modelos Moleculares , Pandemias , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
20.
Biosci Rep ; 40(5)2020 05 29.
Artículo en Inglés | MEDLINE | ID: covidwho-197823

RESUMEN

Since 2002, ß coronaviruses (CoVs) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019). Spike (S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the nCoV2019 takes its course throughout the world, more and more sequence analyses are being done and genome sequences are being deposited in various databases. From India, two clinical isolates have been sequenced and the full genome has been deposited in GenBank. We have performed sequence analyses of the Spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates, one was found to harbor a mutation in its receptor-binding domain (RBD) at position 407. At this site, arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-ß branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor binding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Mutación Puntual , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Betacoronavirus/aislamiento & purificación , Humanos , India , Modelos Moleculares , Pandemias , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
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