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1.
PLoS Pathog ; 16(8): e1008775, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866218

RESUMO

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.


Assuntos
Vírus da Influenza A/metabolismo , Mutação de Sentido Incorreto , Proteínas da Matriz Viral/metabolismo , Células A549 , Substituição de Aminoácidos , Animais , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas da Matriz Viral/genética
2.
J Transl Med ; 18(1): 362, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32967693

RESUMO

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Variação Genética , Pneumonia Viral/virologia , Substituição de Aminoácidos , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , Genoma Viral , Humanos , Itália/epidemiologia , Mutação , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Polimorfismo de Nucleotídeo Único , Pesquisa Médica Translacional , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Cultura de Vírus/métodos , Replicação Viral/genética , Replicação Viral/fisiologia , Sequenciamento Completo do Genoma
3.
Proc Natl Acad Sci U S A ; 117(37): 23165-23173, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868448

RESUMO

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.


Assuntos
Genes Bacterianos/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Substituição de Aminoácidos/genética , Escherichia coli/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Poliproteínas/genética , Proteômica/instrumentação
4.
PLoS One ; 15(8): e0237474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857767

RESUMO

The effective treatment of carbapenemase-producing Klebsiella pneumoniae infection has been limited and required novel potential agents. Due to the novel drug development crisis, using old antimicrobial agents and combination therapy have been highlighted. This study focused on fosfomycin which inhibits cell wall synthesis and has potential activity on Enterobacteriaceae. We evaluated fosfomycin activity against carbapenemase-producing K. pneumoniae and characterized fosfomycin resistance mechanisms. Fosfomycin revealed effective activity against only 31.8% of carbapenemase-producing K. pneumoniae isolates. The major resistance mechanism was FosA3 production. The co-occurrence of FosA3 overexpression with the mutation of glpT (or loss of glpT) and/or uhpT was mediated high-level resistance (MIC>256 mg/L) to fosfomycin. Moreover, fosA3 silenced in sixteen fosfomycin-susceptible isolates and the plasmid carrying fosA3 of these isolates increased 32- to 64-fold of fosfomycin MICs in Escherichia coli DH5α transformants. The in vitro activity of fosfomycin combination with amikacin by checkerboard assay showed synergism and no interaction in six (16.2%) and sixteen isolates (43.3%), respectively. No antagonism of fosfomycin and amikacin was observed. Notably, the silence of aac (6)'-Ib and aphA6 was observed in amikacin-susceptible isolates. Our study suggests that the combination of fosfomycin and amikacin may be insufficient for the treatment of carbapenemase-producing K. pneumoniae isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Amicacina/farmacologia , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , beta-Lactamases/genética
5.
Science ; 369(6508): 1261-1265, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753553

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Engenharia de Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese , Mutação , Peptidil Dipeptidase A/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química
6.
PLoS Pathog ; 16(8): e1008816, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853241

RESUMO

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica
7.
J Theor Biol ; 505: 110425, 2020 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-32735992

RESUMO

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.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Desenho de Fármacos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Betacoronavirus/genética , Sítios de Ligação , Evolução Molecular Direcionada , Humanos , Pandemias , Peptidil Dipeptidase A/uso terapêutico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
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
10.
ACS Nano ; 14(8): 10616-10623, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32806067

RESUMO

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.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Sítios de Ligação/genética , Desenho de Fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
11.
J Transl Med ; 18(1): 321, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831104

RESUMO

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.


Assuntos
Betacoronavirus/fisiologia , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/genética , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Betacoronavirus/metabolismo , Sítios de Ligação/genética , Infecções por Coronavirus/etnologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Predisposição Genética para Doença/etnologia , Variação Genética , Geografia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etnologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
12.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848127

RESUMO

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


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Dobramento de Proteína , Modificação Traducional de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Supressão Genética , Temperatura
13.
Proc Natl Acad Sci U S A ; 117(35): 21274-21280, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817521

RESUMO

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.


Assuntos
Evolução Molecular , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA/biossíntese , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Conformação Proteica , DNA Polimerase Dirigida por RNA/química
14.
PLoS One ; 15(8): e0238121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845893

RESUMO

Variants implicated in childhood epilepsy have been identified in all four voltage-gated sodium channels that initiate action potentials in the central nervous system. Previous research has focused on the functional effects of particular variants within the most studied of these channels (NaV1.1, NaV1.2 and NaV1.6); however, there have been few comparative studies across channels to infer the impact of mutations in patients with epilepsy. Here we compare patterns of variation in patient and public databases to test the hypothesis that regions of known functional significance within voltage-gated sodium (NaV) channels have an increased burden of deleterious variants. We assessed mutational burden in different regions of the Nav channels by (1) performing Fisher exact tests on odds ratios to infer excess variants in domains, segments, and loops of each channel in patient databases versus public "control" databases, and (2) comparing the cumulative distribution of variant sites along DNA sequences of each gene in patient and public databases (i.e., independent of protein structure). Patient variant density was concordant among channels in regions known to play a role in channel function, with statistically significant higher patient variant density in S4-S6 and DIII-DIV and an excess of public variants in SI-S3, DI-DII, DII-DIII. On the other hand, channel-specific patterns of patient burden were found in the NaV1.6 inactivation gate and NaV1.1 S5-S6 linkers, while NaV1.2 and NaV1.6 S4-S5 linkers and S5 segments shared patient variant patterns that contrasted with those in NaV1.1. These different patterns may reflect different roles played by the NaV1.6 inactivation gate in action potential propagation, and by NaV1.1 S5-S6 linkers in loss of function and haploinsufficiency. Interestingly, NaV1.2 and NaV1.6 both lack amino acid substitutions over significantly long stretches in both the patient and public databases suggesting that new mutations in these regions may cause embryonic lethality or a non-epileptic disease phenotype.


Assuntos
Epilepsia/patologia , Ativação do Canal Iônico/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Encéfalo/fisiologia , Epilepsia/genética , Variação Genética/genética , Humanos , Potenciais da Membrana/genética , Mutação/genética , Técnicas de Patch-Clamp , Análise de Sequência de DNA
15.
Mol Pharmacol ; 98(3): 250-266, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817461

RESUMO

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.


Assuntos
Substituição de Aminoácidos , Doença de Parkinson/genética , Receptor A1 de Adenosina/química , Receptor A1 de Adenosina/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Receptor A1 de Adenosina/genética , Termodinâmica
16.
Science ; 369(6510): 1501-1505, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32703906

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


Assuntos
Substituição de Aminoácidos , Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Humanos , Prolina/química , Domínios Proteicos , Estabilidade Proteica , Vacinas Virais/química
17.
BMC Infect Dis ; 20(1): 513, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677899

RESUMO

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.


Assuntos
Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Adulto , África/epidemiologia , Substituição de Aminoácidos/genética , Antimaláricos/uso terapêutico , China/epidemiologia , Estudos de Coortes , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Doença Relacionada a Viagens
18.
BMC Infect Dis ; 20(1): 518, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677920

RESUMO

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.


Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/uso terapêutico , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Substituição de Aminoácidos , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Criança , Ciprofloxacino/uso terapêutico , Análise Mutacional de DNA/métodos , Diarreia/diagnóstico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Humanos , Isoleucina/genética , Testes de Sensibilidade Microbiana , Peru , Treonina/genética
19.
BMC Bioinformatics ; 21(1): 311, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677889

RESUMO

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.


Assuntos
Clonagem Molecular , Biologia Computacional/métodos , Primers do DNA/metabolismo , Poliploidia , Homologia de Sequência , Software , Triticum/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética
20.
PLoS One ; 15(7): e0234792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614850

RESUMO

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.


Assuntos
Proteínas Angiogênicas/biossíntese , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Substituição de Aminoácidos , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Encéfalo/citologia , Proteínas de Transporte/análise , Linhagem da Célula , Epitopos/imunologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Desenvolvimento Muscular , Proteína MyoD/análise , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Coelhos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Sequências Repetitivas de Aminoácidos , Pele/citologia , Especificidade da Espécie , Tatuagem , Adulto Jovem
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