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1.
Biochemistry ; 59(39): 3741-3756, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32931703

RESUMO

The SARS-CoV-2 main protease (Mpro) is essential to viral replication and cleaves highly specific substrate sequences, making it an obvious target for inhibitor design. However, as for any virus, SARS-CoV-2 is subject to constant neutral drift and selection pressure, with new Mpro mutations arising over time. Identification and structural characterization of Mpro variants is thus critical for robust inhibitor design. Here we report sequence analysis, structure predictions, and molecular modeling for seventy-nine Mpro variants, constituting all clinically observed mutations in this protein as of April 29, 2020. Residue substitution is widely distributed, with some tendency toward larger and more hydrophobic residues. Modeling and protein structure network analysis suggest differences in cohesion and active site flexibility, revealing patterns in viral evolution that have relevance for drug discovery.


Assuntos
Betacoronavirus/enzimologia , Betacoronavirus/genética , Modelos Moleculares , Mutação , Proteínas não Estruturais Virais/genética , Domínio Catalítico , Descoberta de Drogas , Evolução Molecular , Humanos , Estrutura Molecular , Filogenia , Inibidores de Proteases/química , Análise de Sequência de Proteína , Proteínas não Estruturais Virais/antagonistas & inibidores
2.
Infect Dis Poverty ; 9(1): 132, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938504

RESUMO

BACKGROUND: Coronavirus disease 2019 (COVID-19) linked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause severe illness and life-threatening pneumonia in humans. The current COVID-19 pandemic demands an effective vaccine to acquire protection against the infection. Therefore, the present study was aimed to design a multiepitope-based subunit vaccine (MESV) against COVID-19. METHODS: Structural proteins (Surface glycoprotein, Envelope protein, and Membrane glycoprotein) of SARS-CoV-2 are responsible for its prime functions. Sequences of proteins were downloaded from GenBank and several immunoinformatics coupled with computational approaches were employed to forecast B- and T- cell epitopes from the SARS-CoV-2 highly antigenic structural proteins to design an effective MESV. RESULTS: Predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (HLA) binding alleles, and collective global population coverage of 88.40%. Taken together, 276 amino acids long MESV was designed by connecting 3 cytotoxic T lymphocytes (CTL), 6 helper T lymphocyte (HTL) and 4 B-cell epitopes with suitable adjuvant and linkers. The MESV construct was non-allergenic, stable, and highly antigenic. Molecular docking showed a stable and high binding affinity of MESV with human pathogenic toll-like receptors-3 (TLR3). Furthermore, in silico immune simulation revealed significant immunogenic response of MESV. Finally, MEV codons were optimized for its in silico cloning into the Escherichia coli K-12 system, to ensure its increased expression. CONCLUSION: The MESV developed in this study is capable of generating immune response against COVID-19. Therefore, if designed MESV further investigated experimentally, it would be an effective vaccine candidate against SARS-CoV-2 to control and prevent COVID-19.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Humanos , Imunogenicidade da Vacina/imunologia , Simulação de Acoplamento Molecular , Pneumonia Viral/imunologia , Análise de Sequência de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Vacinas de Subunidades/química , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinologia/métodos , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Vacinas Virais/química , Vacinas Virais/genética
3.
PLoS Negl Trop Dis ; 14(8): e0008090, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817670

RESUMO

BACKGROUND: Hantaan virus (HTNV; family Hantaviridae, order Bunyavirales) causes hemorrhagic fever with renal syndrome (HFRS), which has raised serious concerns in Eurasia, especially in China, Russia, and South Korea. Previous studies reported genetic diversity and phylogenetic features of HTNV in different parts of China, but the analyses from the holistic perspective are rare. METHODOLOGY AND PRINCIPAL FINDINGS: To better understand HTNV genetic diversity and gene evolution, we analyzed all available complete sequences derived from the small (S) and medium (M) segments with bioinformatic tools. Eleven phylogenetic groups were defined and showed geographic clustering; 42 significant amino acid variant sites were found, and 19 of them were located in immune epitopes; nine recombinant events and eight reassortments with highly divergent sequences were found and analyzed. We found that sequences from Guizhou showed high genetic divergence, contributing to multiple lineages of the phylogenetic tree and also to the recombination and reassortment events. Bayesian stochastic search variable selection analysis revealed that Heilongjiang, Shaanxi, and Guizhou played important roles in HTNV evolution and migration; the virus may originate from Zhejiang Province in the eastern part of China; and the virus population size expanded from the 1980s to 1990s. CONCLUSIONS/SIGNIFICANCE: These findings revealed the original and evolutionary features of HTNV, which will help to illustrate hantavirus epidemic trends, thus aiding in disease control and prevention.


Assuntos
Evolução Molecular , Variação Genética , Vírus Hantaan/genética , Animais , China/epidemiologia , Genoma Viral , Infecções por Hantavirus/epidemiologia , Humanos , Filogenia , RNA Viral/genética , Roedores , Análise de Sequência de Proteína , Musaranhos
4.
Nat Commun ; 11(1): 3784, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728052

RESUMO

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


Assuntos
Bacteriófagos/genética , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Edição de Genes/métodos , Interações Hospedeiro-Parasita/genética , Homologia de Sequência de Aminoácidos
5.
PLoS Negl Trop Dis ; 14(6): e0008323, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559186

RESUMO

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Reações Cruzadas , Malária/imunologia , Plasmodium knowlesi/imunologia , Plasmodium vivax/imunologia , Animais , Humanos , Camundongos , Análise de Sequência de Proteína
6.
Int J Biol Macromol ; 162: 820-837, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32599237

RESUMO

SARS-CoV-2 is the deadly virus behind COVID-19, the disease that went on to ravage the world and caused the biggest pandemic 21st century has witnessed so far. On the face of ongoing death and destruction, the urgent need for the discovery of a vaccine against the virus is paramount. This study resorted to the emerging discipline of immunoinformatics in order to design a multi-epitope mRNA vaccine against the spike glycoprotein of SARS-CoV-2. Various immunoinformatics tools were utilized to predict T and B lymphocyte epitopes. The epitopes were channeled through a filtering pipeline comprised of antigenicity, toxicity, allergenicity, and cytokine inducibility evaluation with the goal of selecting epitopes capable of generating both T and B cell-mediated immune responses. Molecular docking simulation between the epitopes and their corresponding MHC molecules was carried out. 13 epitopes, a highly immunogenic adjuvant, elements for proper sub-cellular trafficking, a secretion booster, and appropriate linkers were combined for constructing the vaccine. The vaccine was found to be antigenic, almost neutral at physiological pH, non-toxic, non-allergenic, capable of generating a robust immune response and had a decent worldwide population coverage. Based on these parameters, this design can be considered a promising choice for a vaccine against SARS-CoV-2.


Assuntos
Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , RNA Mensageiro/imunologia , Vacinas Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Análise de Sequência de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/química , Vacinas Virais/genética
7.
J Med Microbiol ; 69(6): 864-873, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32469301

RESUMO

Introduction. The emergence of SARS-CoV-2 has taken humanity off guard. Following an outbreak of SARS-CoV in 2002, and MERS-CoV about 10 years later, SARS-CoV-2 is the third coronavirus in less than 20 years to cross the species barrier and start spreading by human-to-human transmission. It is the most infectious of the three, currently causing the COVID-19 pandemic. No treatment has been approved for COVID-19. We previously proposed targets that can serve as binding sites for antiviral drugs for multiple coronaviruses, and here we set out to find current drugs that can be repurposed as COVID-19 therapeutics.Aim. To identify drugs against COVID-19, we performed an in silico virtual screen with the US Food and Drug Administration (FDA)-approved drugs targeting the RNA-dependent RNA polymerase (RdRP), a critical enzyme for coronavirus replication.Methodology. Initially, no RdRP structure of SARS-CoV-2 was available. We performed basic sequence and structural analysis to determine if RdRP from SARS-CoV was a suitable replacement. We performed molecular dynamics simulations to generate multiple starting conformations that were used for the in silico virtual screen. During this work, a structure of RdRP from SARS-CoV-2 became available and was also included in the in silico virtual screen.Results. The virtual screen identified several drugs predicted to bind in the conserved RNA tunnel of RdRP, where many of the proposed targets were located. Among these candidates, quinupristin is particularly interesting because it is expected to bind across the RNA tunnel, blocking access from both sides and suggesting that it has the potential to arrest viral replication by preventing viral RNA synthesis. Quinupristin is an antibiotic that has been in clinical use for two decades and is known to cause relatively minor side effects.Conclusion. Quinupristin represents a potential anti-SARS-CoV-2 therapeutic. At present, we have no evidence that this drug is effective against SARS-CoV-2 but expect that the biomedical community will expeditiously follow up on our in silico findings.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Animais , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Betacoronavirus/genética , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Humanos , Conformação Molecular , Pandemias , Filogenia , Pneumonia Viral/virologia , RNA Replicase/efeitos dos fármacos , Rifampina/farmacologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Virginiamicina/análogos & derivados , Virginiamicina/farmacologia , Replicação Viral/efeitos dos fármacos
8.
PLoS Pathog ; 16(5): e1008190, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413071

RESUMO

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.


Assuntos
Leishmania donovani/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cromatina/genética , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Leishmania donovani/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Análise de Sequência de Proteína/métodos
9.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
10.
Microbes Infect ; 22(4-5): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32446902

RESUMO

Envelope protein of coronaviruses is a structural protein existing in both monomeric and homo-pentameric form. It has been related to a multitude of roles including virus infection, replication, dissemination and immune response stimulation. In the present study, we employed an immunoinformatic approach to investigate the major immunogenic domains of the SARS-CoV-2 envelope protein and map them among the homologue proteins of coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Also, when not available, we predicted the envelope protein structural folding and mapped SARS-CoV-2 epitopes. Envelope sequences alignment provides evidence of high sequence homology for some of the investigated virus specimens; while the structural mapping of epitopes resulted in the interesting maintenance of the structural folding and epitope sequence localization also in the envelope proteins scoring a lower alignment score. In line with the One-Health approach, our evidences provide a molecular structural rationale for a potential role of taxonomically related coronaviruses in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.


Assuntos
Betacoronavirus/metabolismo , Biologia Computacional/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas do Envelope Viral/imunologia , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/imunologia , Mapeamento de Epitopos , Regulação Viral da Expressão Gênica , Humanos , Modelos Moleculares , Saúde Única , Pandemias , Filogenia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína
11.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32404529

RESUMO

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a pandemic. Here, we used X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptors allows the definition of residues important for binding. From the 20 amino acids in ACE2 that contact S, up to 7 can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or acquisition of N-glycosylation sites located near the S interface. Of note, pigs and dogs, which are not infected or are not effectively infected and have only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with that from bat coronavirus strain RaTG13 (Bat-CoV-RaTG13) and pangolin coronavirus (Pangolin-CoV) strain hCoV-19/pangolin/Guangdong/1/2019 revealed that the latter contains only one substitution, whereas Bat-CoV-RaTG13 exhibits five. However, ACE2 of pangolin exhibits seven changes relative to human ACE2, and a similar number of substitutions is present in ACE2 of bats, raccoon dogs, and civets, suggesting that SARS-CoV-2 may not be especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-CoV-2.IMPORTANCE SARS-CoV-2 is threatening people worldwide, and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear, and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes, indicating that the species barrier might be low. Exceptions are dogs and especially pigs, which revealed relatively low ACE2 expression levels in the respiratory tract. Monitoring of animals is necessary to prevent the generation of a new coronavirus reservoir. Finally, our analysis also showed that SARS-CoV-2 may not be specifically adapted to any of its putative intermediate hosts.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , Animais , Animais Domésticos , Betacoronavirus/metabolismo , Quirópteros/virologia , Infecções por Coronavirus/metabolismo , Cães , Glicosilação , Interações Hospedeiro-Patógeno , Humanos , Modelos Animais , Pandemias , Animais de Estimação , Pneumonia Viral/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Guaxinins/virologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Suínos , Viverridae/virologia
12.
Nat Commun ; 11(1): 2125, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358559

RESUMO

Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by "negative constraints", which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Drosophila , Proteínas de Drosophila/genética , Evolução Molecular , Estrutura Secundária de Proteína , Análise de Sequência de Proteína
13.
Plant Mol Biol ; 103(4-5): 561-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32405802

RESUMO

KEY MESSAGE: CmHKT1;1 selectively exports Na+ from plant cells. Upon NaCl stress, its expression increased in a salt-tolerant melon cultivar. Overexpression of CmHKT1;1 increased transgenic Arabidopsis salt tolerance through improved K+/Na+ balance. High-affinity K+ transporters (HKTs) are thought to be involved in reducing Na+ in plant shoots under salt stress and modulating salt tolerance, but their function in a moderately salt-tolerant species of melon (Cucumis melo L.) remains unclear. In this study, a Na+ transporter gene, CmHKT1;1 (GenBank accession number: MK986658), was isolated from melons based on genome data. The transcript of CmHKT1;1 was relatively more abundant in roots than in stems or leaves from melon seedlings. The tobacco transient expression system showed that CmHKT1;1 was plasma-membrane localized. Upon salt stress, CmHKT1;1 expression was more strongly upregulated in a salt-tolerant melon cultivar, 'Bingxuecui' (BXC) compared with a salt-sensitive cultivar, 'Yulu' (YL). Electrophysiological evidence demonstrated that CmHKT1;1 only transported Na+, rather than K+, when expressed in Xenopus laevis oocytes. Overexpression of CmHKT1;1 increased salt sensitivity in Saccharomyces cerevisiae and salt tolerance in Arabidopsis thaliana. Under NaCl treatments, transgenic Arabidopsis plants accumulated significantly lower concentrations of Na+ in shoots than wild type plants and showed a better K+/Na+ balance, leading to better Fv/Fm, root length, biomass, and enhanced plant growth. The CmHKT1;1 gene may serve as a useful candidate for improving crop salt tolerance.


Assuntos
Arabidopsis/metabolismo , Cucumis melo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Clorofila/análise , Clonagem Molecular , Cucumis melo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal , Plântula/genética , Plântula/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Simportadores/genética , Simportadores/metabolismo , Tabaco/genética , Tabaco/metabolismo
14.
Nucleic Acids Res ; 48(W1): W154-W161, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32352516

RESUMO

The separation of deleterious from benign mutations remains a key challenge in the interpretation of genomic data. Computational methods used to sort mutations based on their potential deleteriousness rely largely on conservation measures derived from sequence alignments. Here, we introduce LIST-S2, a successor to our previously developed approach LIST, which aims to exploit local sequence identity and taxonomy distances in quantifying the conservation of human protein sequences. Unlike its predecessor, LIST-S2 is not limited to human sequences but can assess conservation and make predictions for sequences from any organism. Moreover, we provide a web-tool and downloadable software to compute and visualize the deleteriousness of mutations in user-provided sequences. This web-tool contains an HTML interface and a RESTful API to submit and manage sequences as well as a browsable set of precomputed predictions for a large number of UniProtKB protein sequences of common taxa. LIST-S2 is available at: https://list-s2.msl.ubc.ca/.


Assuntos
Mutação de Sentido Incorreto , Software , Animais , Mutação em Linhagem Germinativa , Humanos , Neoplasias/genética , Análise de Sequência de Proteína
15.
Nucleic Acids Res ; 48(W1): W104-W109, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32392342

RESUMO

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochemical characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochemical characterization based on rational and robust analysis of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab experiments. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a soluble form in Escherichia coli. The solubility prediction employs the in-house SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step analysis, sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page. EnzymeMiner is a universal tool applicable to any enzyme family that provides an interactive and easy-to-use web interface freely available at https://loschmidt.chemi.muni.cz/enzymeminer/.


Assuntos
Enzimas/química , Software , Biocatálise , Estabilidade Enzimática , Enzimas/metabolismo , Hidrolases/química , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Solubilidade
16.
Nucleic Acids Res ; 48(W1): W110-W115, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32406917

RESUMO

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.


Assuntos
Metabolismo dos Carboidratos , Enzimas/química , Enzimas/genética , Anotação de Sequência Molecular , Software , Algoritmos , Enzimas/classificação , Enzimas/metabolismo , Internet , Peptídeos/química , Análise de Sequência de DNA , Análise de Sequência de Proteína
17.
Nucleic Acids Res ; 48(W1): W77-W84, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32421769

RESUMO

Low complexity regions (LCRs) in protein sequences are characterized by a less diverse amino acid composition compared to typically observed sequence diversity. Recent studies have shown that LCRs may co-occur with intrinsically disordered regions, are highly conserved in many organisms, and often play important roles in protein functions and in diseases. In previous decades, several methods have been developed to identify regions with LCRs or amino acid bias, but most of them as stand-alone applications and currently there is no web-based tool which allows users to explore LCRs in protein sequences with additional functional annotations. We aim to fill this gap by providing PlaToLoCo - PLAtform of TOols for LOw COmplexity-a meta-server that integrates and collects the output of five different state-of-the-art tools for discovering LCRs and provides functional annotations such as domain detection, transmembrane segment prediction, and calculation of amino acid frequencies. In addition, the union or intersection of the results of the search on a query sequence can be obtained. By developing the PlaToLoCo meta-server, we provide the community with a fast and easily accessible tool for the analysis of LCRs with additional information included to aid the interpretation of the results. The PlaToLoCo platform is available at: http://platoloco.aei.polsl.pl/.


Assuntos
Proteínas/química , Software , Aminoácidos/análise , Gráficos por Computador , Humanos , Proteínas de Membrana/química , Anotação de Sequência Molecular , Domínios Proteicos , Análise de Sequência de Proteína
18.
Nucleic Acids Res ; 48(W1): W348-W357, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32459325

RESUMO

Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR-Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR-Cas machineries for the development of new CRISPR-Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.


Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Software , Proteínas Virais/química , Gráficos por Computador , Aprendizado de Máquina , Análise de Sequência de Proteína
19.
Nucleic Acids Res ; 48(W1): W25-W30, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32383764

RESUMO

The accurate and reliable prediction of the 3D structures of proteins and their assemblies remains difficult even though the number of solved structures soars and prediction techniques improve. In this study, a free and open access web server, AWSEM-Suite, whose goal is to predict monomeric protein tertiary structures from sequence is described. The model underlying the server's predictions is a coarse-grained protein force field which has its roots in neural network ideas that has been optimized using energy landscape theory. Employing physically motivated potentials and knowledge-based local structure biasing terms, the addition of homologous template and co-evolutionary restraints to AWSEM-Suite greatly improves the predictive power of pure AWSEM structure prediction. From the independent evaluation metrics released in the CASP13 experiment, AWSEM-Suite proves to be a reasonably accurate algorithm for free modeling, standing at the eighth position in the free modeling category of CASP13. The AWSEM-Suite server also features a front end with a user-friendly interface. The AWSEM-Suite server is a powerful tool for predicting monomeric protein tertiary structures that is most useful when a suitable structure template is not available. The AWSEM-Suite server is freely available at: https://awsem.rice.edu.


Assuntos
Estrutura Terciária de Proteína , Software , Algoritmos , Evolução Molecular , Dobramento de Proteína , Análise de Sequência de Proteína , Homologia Estrutural de Proteína
20.
Int J Antimicrob Agents ; 55(5): 105960, 2020 May.
Artigo em Inglês | MEDLINE | ID: covidwho-41048

RESUMO

The recent emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is responsible for a worldwide pandemic. Given the global health emergency, drug repositioning is the most reliable option to design an efficient therapy for infected patients without delay. The first step of the viral replication cycle [i.e. attachment to the surface of respiratory cells, mediated by the spike (S) viral protein] offers several potential therapeutic targets. The S protein uses the angiotension-converting enzyme-2 (ACE-2) receptor for entry, but also sialic acids linked to host cell surface gangliosides. Using a combination of structural and molecular modelling approaches, this study showed that chloroquine (CLQ), one of the drugs currently under investigation for SARS-CoV-2 treatment, binds sialic acids and gangliosides with high affinity. A new type of ganglioside-binding domain at the tip of the N-terminal domain of the SARS-CoV-2 S protein was identified. This domain (111-158), which is fully conserved among clinical isolates worldwide, may improve attachment of the virus to lipid rafts and facilitate contact with the ACE-2 receptor. This study showed that, in the presence of CLQ [or its more active derivative, hydroxychloroquine (CLQ-OH)], the viral S protein is no longer able to bind gangliosides. The identification of this new mechanism of action of CLQ and CLQ-OH supports the use of these repositioned drugs to cure patients infected with SARS-CoV-2. The in-silico approaches used in this study might also be used to assess the efficiency of a broad range of repositioned and/or innovative drug candidates before clinical evaluation.


Assuntos
Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Hidroxicloroquina/farmacologia , Pneumonia Viral/tratamento farmacológico , Sequência de Aminoácidos , Betacoronavirus/química , Cloroquina/química , Cloroquina/uso terapêutico , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/uso terapêutico , Modelos Moleculares , Terapia de Alvo Molecular , Pandemias , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Glicoproteína da Espícula de Coronavírus/química
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