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1.
Front Immunol ; 12: 693775, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484190

RESUMO

Small number of SARS-CoV-2 epidemic lineages did not efficiently exhibit a neutralization profile, while single amino acid mutation in the spike protein has not been confirmed in altering viral antigenicity resulting in immune escape. To identify crucial mutations in spike protein that escape humoral immune response, we evaluated the cross-neutralization of convalescent plasmas and RBD-specific monoclonal antibodies (mAbs) against various spike protein-based pseudoviruses. Three of 24 SARS-CoV-2 pseudoviruses containing different mutations in spike protein, including D614G, A475V, and E484Q, consistently showed an altered sensitivity to neutralization by convalescent plasmas. A475V and E484Q mutants are highly resistant to neutralization by mAb B38 and 2-4, suggesting that some crucial mutations in spike protein might evolve SARS-CoV-2 variants capable of escaping humoral immune response.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/imunologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Anticorpos Neutralizantes/imunologia , Convalescença , Humanos , Evasão da Resposta Imune , Imunidade Humoral , Testes de Neutralização , Ligação Proteica
2.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445264

RESUMO

The multidrug efflux transporter ABCB1 is clinically important for drug absorption and distribution and can be a determinant of chemotherapy failure. Recent structure data shows that three glutamines donate hydrogen bonds to coordinate taxol in the drug binding pocket. This is consistent with earlier drug structure-activity relationships that implicated the importance of hydrogen bonds in drug recognition by ABCB1. By replacing the glutamines with alanines we have tested whether any, or all, of Gln347, Gln725, and Gln990 are important for the transport of three different drug classes. Flow cytometric transport assays show that Q347A and Q990A act synergistically to reduce transport of Calcein-AM, BODIPY-verapamil, and OREGON GREEN-taxol bisacetate but the magnitude of the effect was dependent on the test drug and no combination of mutations completely abrogated function. Surprisingly, Q725A mutants generally improved transport of Calcein-AM and BODIPY-verapamil, suggesting that engagement of the wild-type Gln725 in a hydrogen bond is inhibitory for the transport mechanism. To test transport of unmodified taxol, stable expression of Q347/725A and the triple mutant was engineered and shown to confer equivalent resistance to the drug as the wild-type transporter, further indicating that none of these potential hydrogen bonds between transporter and transport substrate are critical for the function of ABCB1. The implications of the data for plasticity of the drug binding pocket are discussed.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Paclitaxel/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Resistencia a Medicamentos Antineoplásicos/genética , Glutamina/genética , Glutamina/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto
3.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445292

RESUMO

The genes involved in implantation and placentation are tightly regulated to ensure a healthy pregnancy. The endoplasmic reticulum aminopeptidase 2 (ERAP2) gene is associated with preeclampsia (PE). Our studies have determined that an isoform of ERAP2-arginine (N), expressed in trophoblast cells (TC), significantly activates immune cells, and ERAP2N-expressing TCs are preferentially killed by both cytotoxic T lymphocytes (CTLs) and Natural Killer cells (NKCs). To understand the cause of this phenomenon, we surveyed differentially expressed genes (DEGs) between ERAP2N expressing and non-expressing TCs. Our RNAseq data revealed 581 total DEGs between the two groups. 289 genes were up-regulated, and 292 genes were down-regulated. Interestingly, most of the down-regulated genes of significance were pro-survival genes that play a crucial role in cell survival (LDHA, EGLN1, HLA-C, ITGB5, WNT7A, FN1). However, the down-regulation of these genes in ERAP2N-expressing TCs translates into a propensity for cell death. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 64 DEGs were significantly enriched in nine pathways, including "Protein processing in endoplasmic reticulum" and "Antigen processing and presentation", suggesting that the genes may be associated with peptide processes involved in immune recognition during the reproductive cycle.


Assuntos
Aminopeptidases/genética , Morte Celular/genética , Trofoblastos/metabolismo , Substituição de Aminoácidos/genética , Aminopeptidases/metabolismo , Arginina/genética , Células Cultivadas , Citotoxicidade Imunológica/genética , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologia , Trofoblastos/fisiologia , Regulação para Cima/genética
4.
BMC Plant Biol ; 21(1): 373, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388969

RESUMO

BACKGROUND: Flowering is an important inflection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. RESULTS: In this study, a stable early-bolting mutant, ebm3, was identified in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line 'FT'. Compared with 'FT', ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modified MutMap sequencing, genotyping analyses and allelism test provide strong evidence that BrEBM3 (BraA04g017190.3 C), encoding the histone methyltransferase CURLY LEAF (CLF), was the strongly candidate gene of the emb3. A C to T base substitution in the 14th exon of BrEBM3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3-9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specific lysine methylation in histones. Tissue-specific expression analysis showed that BrEBM3 was highly expressed in the flower and bud. Promoter activity assay confirmed that BrEBM3 promoter was active in inflorescences. Subcellular localization analysis revealed that BrEBM3 localized in the nucleus. Transcriptomic studies supported that BrEBM3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early flowering. CONCLUSIONS: Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3 C, encoding the histone methyltransferase CLF. These results improve our knowledge of the genetic and genomic resources of bolting and flowering, and may be beneficial to the genetic improvement of Chinese cabbage.


Assuntos
Substituição de Aminoácidos , Brassica rapa/enzimologia , Histona Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Brassica rapa/genética , Brassica rapa/crescimento & desenvolvimento , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Histona Metiltransferases/química , Histona Metiltransferases/genética , Mutação , Proteínas de Plantas/genética , Transcriptoma
5.
PLoS Comput Biol ; 17(8): e1009284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34347784

RESUMO

Modeling the impact of amino acid mutations on protein-protein interaction plays a crucial role in protein engineering and drug design. In this study, we develop GeoPPI, a novel structure-based deep-learning framework to predict the change of binding affinity upon mutations. Based on the three-dimensional structure of a protein, GeoPPI first learns a geometric representation that encodes topology features of the protein structure via a self-supervised learning scheme. These representations are then used as features for training gradient-boosting trees to predict the changes of protein-protein binding affinity upon mutations. We find that GeoPPI is able to learn meaningful features that characterize interactions between atoms in protein structures. In addition, through extensive experiments, we show that GeoPPI achieves new state-of-the-art performance in predicting the binding affinity changes upon both single- and multi-point mutations on six benchmark datasets. Moreover, we show that GeoPPI can accurately estimate the difference of binding affinities between a few recently identified SARS-CoV-2 antibodies and the receptor-binding domain (RBD) of the S protein. These results demonstrate the potential of GeoPPI as a powerful and useful computational tool in protein design and engineering. Our code and datasets are available at: https://github.com/Liuxg16/GeoPPI.


Assuntos
Substituição de Aminoácidos , Modelos Químicos , Proteínas/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas/química , Proteínas/genética
6.
PLoS Pathog ; 17(8): e1009772, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34352039

RESUMO

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.


Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Linhagem Celular , Humanos , Evasão da Resposta Imune , Mutação/genética , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
Viruses ; 13(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34452356

RESUMO

Here, we report on the increasing frequency of the SARS-CoV-2 lineage A.27 in Germany during the first months of 2021. Genomic surveillance identified 710 A.27 genomes in Germany as of 2 May 2021, with a vast majority identified in laboratories from a single German state (Baden-Wuerttemberg, n = 572; 80.5%). Baden-Wuerttemberg is located near the border with France, from where most A.27 sequences were entered into public databases until May 2021. The first appearance of this lineage based on sequencing in a laboratory in Baden-Wuerttemberg can be dated to early January '21. From then on, the relative abundance of A.27 increased until the end of February but has since declined-meanwhile, the abundance of B.1.1.7 increased in the region. The A.27 lineage shows a mutational pattern typical of VOIs/VOCs, including an accumulation of amino acid substitutions in the Spike glycoprotein. Among those, L18F, L452R and N501Y are located in the epitope regions of the N-terminal- (NTD) or receptor binding domain (RBD) and have been suggested to result in immune escape and higher transmissibility. In addition, A.27 does not show the D614G mutation typical for all VOIs/VOCs from the B lineage. Overall, A.27 should continue to be monitored nationally and internationally, even though the observed trend in Germany was initially displaced by B.1.1.7 (Alpha), while now B.1.617.2 (Delta) is on the rise.


Assuntos
COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Substituição de Aminoácidos , COVID-19/epidemiologia , França/epidemiologia , Genoma Viral , Alemanha/epidemiologia , Humanos , Mutação , Filogenia , SARS-CoV-2/classificação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
Nucleic Acids Res ; 49(15): 8796-8810, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379778

RESUMO

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Assuntos
Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Mutação , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Proteínas Virais/genética
9.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360790

RESUMO

Genetic variations have a multitude of effects on proteins. A substantial number of variations affect protein-solvent interactions, either aggregation or solubility. Aggregation is often related to structural alterations, whereas solubilizable proteins in the solid phase can be made again soluble by dilution. Solubility is a central protein property and when reduced can lead to diseases. We developed a prediction method, PON-Sol2, to identify amino acid substitutions that increase, decrease, or have no effect on the protein solubility. The method is a machine learning tool utilizing gradient boosting algorithm and was trained on a large dataset of variants with different outcomes after the selection of features among a large number of tested properties. The method is fast and has high performance. The normalized correct prediction rate for three states is 0.656, and the normalized GC2 score is 0.312 in 10-fold cross-validation. The corresponding numbers in the blind test were 0.545 and 0.157. The performance was superior in comparison to previous methods. The PON-Sol2 predictor is freely available. It can be used to predict the solubility effects of variants for any organism, even in large-scale projects.


Assuntos
Substituição de Aminoácidos , Análise de Sequência de Proteína , Software , Valor Preditivo dos Testes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade
10.
Front Immunol ; 12: 719115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34367187

RESUMO

Introduction: Loss-of-function TLR7 variants have been recently reported in a small number of males to underlie strong predisposition to severe COVID-19. We aimed to determine the presence of these rare variants in young men with severe COVID-19. Methods: We prospectively studied males between 18 and 50 years-old without predisposing comorbidities that required at least high-flow nasal oxygen to treat COVID-19. The coding region of TLR7 was sequenced to assess the presence of potentially deleterious variants. Results: TLR7 missense variants were identified in two out of 14 patients (14.3%). Overall, the median age was 38 (IQR 30-45) years. Both variants were not previously reported in population control databases and were predicted to be damaging by in silico predictors. In a 30-year-old patient a maternally inherited variant [c.644A>G; p.(Asn215Ser)] was identified, co-segregating in his 27-year-old brother who also contracted severe COVID-19. A second variant [c.2797T>C; p.(Trp933Arg)] was found in a 28-year-old patient, co-segregating in his 24-year-old brother who developed mild COVID-19. Functional testing of this variant revealed decreased type I and II interferon responses in peripheral mononuclear blood cells upon stimulation with the TLR7 agonist imiquimod, confirming a loss-of-function effect. Conclusions: This study supports a rationale for the genetic screening for TLR7 variants in young men with severe COVID-19 in the absence of other relevant risk factors. A diagnosis of TLR7 deficiency could not only inform on treatment options for the patient, but also enables pre-symptomatic testing of at-risk male relatives with the possibility of instituting early preventive and therapeutic interventions.


Assuntos
COVID-19/genética , Mutação de Sentido Incorreto , SARS-CoV-2 , Receptor 7 Toll-Like/genética , Adulto , Substituição de Aminoácidos , COVID-19/imunologia , COVID-19/patologia , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Índice de Gravidade de Doença , Receptor 7 Toll-Like/imunologia
12.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34417349

RESUMO

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/farmacologia , Sítios de Ligação , COVID-19/genética , COVID-19/virologia , Chlorocebus aethiops , Convalescença , Expressão Gênica , Humanos , Evasão da Resposta Imune , Soros Imunes/química , Modelos Moleculares , Mutação , Testes de Neutralização , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
13.
Arch Virol ; 166(9): 2461-2468, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34212242

RESUMO

Bovine coronavirus (BCoV) can be spread by animal activity. Although cattle farming is widespread in Turkey, there are few studies of BCoV. The aim of this study was to evaluate the current situation regarding BCoV in Turkey. This is the first study reporting the full-length nucleotide sequences of BCoV spike (S) genes in Turkey. Samples were collected from 119 cattle with clinical signs of respiratory (n = 78) or digestive tract (n = 41) infection on different farms located across widely separated provinces in Turkey. The samples were screened for BCoV using RT-nested PCR targeting the N gene, which identified BCoV in 35 samples (9 faeces and 26 nasal discharge). RT-PCR analysis of the S gene produced partial/full-length S gene sequences from 11 samples (8 faeces and 3 nasal discharge samples). A phylogenetic tree of the S gene sequences was made to analyze the genetic relationships among BCoVs from Turkey and other countries. The results showed that the local strains present in faeces and nasal discharge samples had many different amino acid changes. Some of these changes were shown in previous studies to be critical for tropism. This study provides new data on BCoV in Turkey that will be valuable in designing effective vaccine approaches and control strategies.


Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Coronavirus/veterinária , Coronavirus Bovino/genética , Diarreia/veterinária , RNA Viral/genética , Infecções Respiratórias/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Agricultura , Substituição de Aminoácidos , Animais , Bovinos , Doenças dos Bovinos/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavirus Bovino/classificação , Diarreia/epidemiologia , Diarreia/virologia , Monitoramento Epidemiológico/veterinária , Evolução Molecular , Fezes/virologia , Humanos , Mutação , Cavidade Nasal/virologia , Filogenia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Turquia/epidemiologia
14.
Nat Commun ; 12(1): 4582, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321470

RESUMO

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.


Assuntos
Ciclo Celular/fisiologia , HIV-1/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Sumoilação/fisiologia , Substituição de Aminoácidos , Células HEK293 , Infecções por HIV/virologia , Humanos , Lisina , Mutação , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/química , Células U937
15.
Fish Shellfish Immunol ; 116: 84-90, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34214656

RESUMO

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.


Assuntos
Septicemia Hemorrágica Viral , Novirhabdovirus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Peixes , Expressão Gênica/imunologia , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/imunologia , Estabilidade Proteica
16.
Cell ; 184(17): 4392-4400.e4, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34289344

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Linhagem Celular , Quirópteros/virologia , Chlorocebus aethiops , Reservatórios de Doenças , Evolução Molecular , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
17.
Cell Rep ; 36(2): 109364, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34214467

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.


Assuntos
COVID-19/imunologia , COVID-19/metabolismo , Receptores Virais , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteômica , Receptores Virais/metabolismo , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Internalização do Vírus , Replicação Viral
18.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208896

RESUMO

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.


Assuntos
Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/genética , Neoplasias Pulmonares/genética , Timidina/genética , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Células HCT116 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Espectroscopia de Ressonância Magnética
19.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205742

RESUMO

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26-38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people's view on inhibitor discovery for the checkpoint pair.


Assuntos
Inibidores de Checkpoint Imunológico , Receptores Imunológicos/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Substituição de Aminoácidos , Evolução Biológica , Simulação por Computador , Descoberta de Drogas , Simulação de Acoplamento Molecular
20.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210092

RESUMO

Brain-derived neurotrophic factor (BDNF) is a pleiotropic neuronal growth and survival factor that is indispensable in the brain, as well as in multiple other tissues and organs, including the cardiovascular system. In approximately 30% of the general population, BDNF harbors a nonsynonymous single nucleotide polymorphism that may be associated with cardiometabolic disorders, coronary artery disease, and Duchenne muscular dystrophy cardiomyopathy. We recently showed that transgenic mice with the human BDNF rs6265 polymorphism (Val66Met) exhibit altered cardiac function, and that cardiomyocytes isolated from these mice are also less contractile. To identify the underlying mechanisms involved, we compared cardiac function by echocardiography and performed deep sequencing of RNA extracted from whole hearts of all three genotypes (Val/Val, Val/Met, and Met/Met) of both male and female Val66Met mice. We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including increased systolic (26.8%, p = 0.047) and diastolic diameters (14.9%, p = 0.022), increased systolic (57.9%, p = 0.039) and diastolic volumes (32.7%, p = 0.026), and increased stroke volume (25.9%, p = 0.033), with preserved ejection fraction and fractional shortening. Both males and females exhibited lower heart rates, but this change was more pronounced in female mice than in males. Consistent with phenotypic observations, the gene encoding SERCA2 (Atp2a2) was reduced in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin (Tcap) and upregulation of BNP (Nppb), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Substituição de Aminoácidos , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Expressão Gênica , Masculino , Metionina/genética , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Caracteres Sexuais , Valina/genética , Função Ventricular/genética , Função Ventricular/fisiologia
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