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1.
Biomol NMR Assign ; 15(1): 219-227, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33660218

RESUMO

The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.


Assuntos
/química , Espectroscopia de Ressonância Magnética , /química , Isótopos de Carbono , Biologia Computacional , Hidrogênio , Isótopos de Nitrogênio , Fosfoproteínas/química , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína
2.
J Mol Biol ; 433(8): 166875, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33556408

RESUMO

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.


Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , /metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli , Humanos , Mutação , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Viral/metabolismo , Especificidade por Substrato
3.
Nucleic Acids Res ; 49(5): 2959-2972, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619523

RESUMO

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.


Assuntos
DNA/química , Proteínas Nucleares/química , Fosfoproteínas/química , Cristalografia por Raios X , DNA/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
4.
Viruses ; 13(2)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540713

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.


Assuntos
Antivirais/farmacologia , Olea , Álcool Feniletílico/análogos & derivados , Extratos Vegetais/farmacologia , /efeitos dos fármacos , Administração Tópica , Animais , Antivirais/química , Carboidratos/química , Chlorocebus aethiops , Genoma Viral/efeitos dos fármacos , Glicosilação , Testes de Sensibilidade Microbiana , Álcool Feniletílico/administração & dosagem , Álcool Feniletílico/farmacologia , Fosfoproteínas/química , Extratos Vegetais/química , /fisiologia , Creme para a Pele , Glicoproteína da Espícula de Coronavírus/química , Células Vero , Inativação de Vírus/efeitos dos fármacos
5.
Viruses ; 13(2)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557213

RESUMO

Monitoring acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity and emerging mutations in this ongoing pandemic is crucial for understanding its evolution and assuring the performance of diagnostic tests, vaccines, and therapies against coronavirus disease (COVID-19). This study reports on the amino acid (aa) conservation degree and the global and regional temporal evolution by epidemiological week for each residue of the following four structural SARS-CoV-2 proteins: spike, envelope, membrane, and nucleocapsid. All, 105,276 worldwide SARS-CoV-2 complete and partial sequences from 117 countries available in the Global Initiative on Sharing All Influenza Data (GISAID) from 29 December 2019 to 12 September 2020 were downloaded and processed using an in-house bioinformatics tool. Despite the extremely high conservation of SARS-CoV-2 structural proteins (>99%), all presented aa changes, i.e., 142 aa changes in 65 of the 75 envelope aa, 291 aa changes in 165 of the 222 membrane aa, 890 aa changes in 359 of the 419 nucleocapsid aa, and 2671 changes in 1132 of the 1273 spike aa. Mutations evolution differed across geographic regions and epidemiological weeks (epiweeks). The most prevalent aa changes were D614G (81.5%) in the spike protein, followed by the R203K and G204R combination (37%) in the nucleocapsid protein. The presented data provide insight into the genetic variability of SARS-CoV-2 structural proteins during the pandemic and highlights local and worldwide emerging aa changes of interest for further SARS-CoV-2 structural and functional analysis.


Assuntos
/virologia , /genética , Evolução Molecular , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Substituição de Aminoácidos , /química , Variação Genética , Genoma Viral , Humanos , Mutação , Pandemias , Fosfoproteínas/química , Fosfoproteínas/genética , Glicoproteína da Espícula de Coronavírus/química , Proteínas da Matriz Viral/química
6.
PLoS One ; 16(1): e0238665, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497392

RESUMO

This study aims to highlight SARS-COV-2 mutations which are associated with increased or decreased viral virulence. We utilize genetic data from all strains available from GISAID and countries' regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of specific mutations can be obtained from calculating their frequencies across viral strains. By applying modelling approaches, we provide additional information that is not evident from standard statistics or mutation frequencies alone. We therefore, propose a more precise way of selecting informative mutations. We highlight two interesting mutations found in genes N (P13L) and ORF3a (Q57H). The former appears to be significantly associated with decreased deaths and cases per million according to our models, while the latter shows an opposing association with decreased deaths and increased cases per million. Moreover, protein structure prediction tools show that the mutations infer conformational changes to the protein that significantly alter its structure when compared to the reference protein.


Assuntos
/virologia , /genética , /genética , /transmissão , Sistemas de Informação Geográfica , Humanos , Modelos Lineares , Mutação , Pandemias , Fosfoproteínas/química , Fosfoproteínas/genética , Filogenia , Polimorfismo de Nucleotídeo Único , /química
7.
PLoS One ; 16(1): e0245382, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497420

RESUMO

Antibody responses are important in the control of viral respiratory infection in the human host. What is not clear for SARS-CoV-2 is how rapidly this response occurs, or when antibodies with protective capability evolve. Hence, defining the events of SARS-CoV-2 seroconversion and the time frame for the development of antibodies with protective potential may help to explain the different clinical presentations of COVID-19. Furthermore, accurate descriptions of seroconversion are needed to inform the best use of serological assays for diagnostic testing and serosurveillance studies. Here, we describe the humoral responses in a cohort of hospitalised COVID-19 patients (n = 19) shortly following the onset of symptoms. Commercial and 'in-house' serological assays were used to measure IgG antibodies against different SARS-CoV-2 structural antigens-Spike (S) S1 sub-unit and Nucleocapsid protein (NP)-and to assess the potential for virus neutralisation mediated specifically by inhibition of binding between the viral attachment protein (S protein) and cognate receptor (ACE-2). Antibody response kinetics varied amongst the cohort, with patients seroconverting within 1 week, between 1-2 weeks, or after 2 weeks, following symptom onset. Anti-NP IgG responses were generally detected earlier, but reached maximum levels slower, than anti-S1 IgG responses. The earliest IgG antibodies produced by all patients included those that recognised the S protein receptor-binding domain (RBD) and were capable of inhibiting binding to ACE-2. These data revealed events and patterns of SARS-CoV-2 seroconversion that may be important predictors of the outcome of infection and guide the delivery of clinical services in the COVID-19 response.


Assuntos
Anticorpos Neutralizantes/imunologia , /imunologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , Estudos de Coortes , Feminino , Hospitalização , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Soroconversão , Glicoproteína da Espícula de Coronavírus/química , País de Gales
8.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33443088

RESUMO

Developing optimal T-cell response assays to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is critical for measuring the duration of immunity to this disease and assessing the efficacy of vaccine candidates. These assays need to target conserved regions of SARS-CoV-2 global variants and avoid cross-reactivity to seasonal human coronaviruses. To contribute to this effort, we employed an in silico immunoinformatics analysis pipeline to identify immunogenic peptides resulting from conserved and highly networked regions with topological importance from the SARS-CoV-2 nucleocapsid and spike proteins. A total of 57 highly networked T-cell epitopes that are conserved across geographic viral variants were identified from these viral proteins, with a binding potential to diverse HLA alleles and 80 to 100% global population coverage. Importantly, 18 of these T-cell epitope derived peptides had limited homology to seasonal human coronaviruses making them promising candidates for SARS-CoV-2-specific T-cell immunity assays. Moreover, two of the NC-derived peptides elicited effector/polyfunctional responses of CD8+ T cells derived from SARS-CoV-2 convalescent patients.IMPORTANCE The development of specific and validated immunologic tools is critical for understanding the level and duration of the cellular response induced by SARS-CoV-2 infection and/or vaccines against this novel coronavirus disease. To contribute to this effort, we employed an immunoinformatics analysis pipeline to define 57 SARS-CoV-2 immunogenic peptides within topologically important regions of the nucleocapsid (NC) and spike (S) proteins that will be effective for detecting cellular immune responses in 80 to 100% of the global population. Our immunoinformatics analysis revealed that 18 of these peptides had limited homology to circulating seasonal human coronaviruses and therefore are promising candidates for distinguishing SARS-CoV-2-specific immune responses from pre-existing coronavirus immunity. Importantly, CD8+ T cells derived from SARS-CoV-2 survivors exhibited polyfunctional effector responses to two novel NC-derived peptides identified as HLA-binders. These studies provide a proof of concept that our immunoinformatics analysis pipeline identifies novel immunogens which can elicit polyfunctional SARS-CoV-2-specific T-cell responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Apresentação do Antígeno , /imunologia , Biologia Computacional , Coronavirus/classificação , Coronavirus/imunologia , /genética , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Antígenos HLA/imunologia , Humanos , Imunidade Celular , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Ligação Proteica , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
9.
Biochem Biophys Res Commun ; 541: 50-55, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477032

RESUMO

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic.


Assuntos
Trifosfato de Adenosina/metabolismo , Extração Líquido-Líquido , RNA Viral/metabolismo , /metabolismo , Trifosfato de Adenosina/química , Sítios de Ligação , /genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Viral/química , RNA Viral/genética , Motivos de Ligação ao RNA/genética , /química
10.
Virology ; 555: 35-43, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33450669

RESUMO

The pandemic of COVID-19 is still ongoing, and many studies on serum antibodies have been reported, however, there are few studies about asymptomatic and mild patients. In this study, we enrolled 44 COVID-19 patients with relatively mild disease and 48 pre-pandemic controls. We measured serum antibodies against extracellular domain, S1 domain, and receptor-binding domain of Spike and N protein, examined neutralization titers by authentic virus neutralization assay and newly-developed bead/cell-based Spike-ACE2 inhibition assay, and compared them with clinical features. Most of these antibodies, including neutralizing titers, were mutually correlated, and the production of antibodies were associated with low Ct values of PCR test, disease severity, symptoms especially pneumonia, lymphopenia, and serological test including CRP, LD, D-dimer, and procalcitonin. Notably, 87.5% of asymptomatic and 23.5% of mild patients did not have antibody against SARS-CoV-2. Our results revealed the inadequate acquisition of humoral immunity in patients with asymptomatic and mild COVID-19 patients.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções Assintomáticas , /fisiopatologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Japão , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Fosfoproteínas/imunologia , Domínios Proteicos , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Exp Eye Res ; 203: 108433, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33400927

RESUMO

Although severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection have emerged globally, findings related to ocular involvement and reported cases are quite limited. Immune reactions against viral infections are closely related to viral and host proteins sequence similarity. Molecular Mimicry has been described for many different viruses; sequence similarities of viral and human tissue proteins may trigger autoimmune reactions after viral infections due to similarities between viral and human structures. With this study, we aimed to investigate the protein sequence similarity of SARS CoV-2 with retinal proteins and retinal pigment epithelium (RPE) surface proteins. Retinal proteins involved in autoimmune retinopathy and retinal pigment epithelium surface transport proteins were analyzed in order to infer their structural similarity to surface glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), envelope protein (E), ORF1ab polyprotein (orf1ab) proteins of SARS CoV-2. Protein similarity comparisons, 3D protein structure prediction, T cell epitopes-MHC binding prediction, B cell epitopes-MHC binding prediction and the evaluation of the antigenicity of peptides assessments were performed. The protein sequence analysis was made using the Pairwise Sequence Alignment and the LALIGN program. 3D protein structure estimates were made using Swiss Model with default settings and analyzed with TM-align web server. T-cell epitope identification was performed using the Immune Epitope Database and Analysis (IEDB) resource Tepitool. B cell epitopes based on sequence characteristics of the antigen was performed using amino acid scales and HMMs with the BepiPred 2.0 web server. The predicted peptides/epitopes in terms of antigenicity were examined using the default settings with the VaxiJen v2.0 server. Analyses showed that, there is a meaningful similarities between 6 retinal pigment epithelium surface transport proteins (MRP-4, MRP-5, RFC1, SNAT7, TAUT and MATE) and the SARS CoV-2 E protein. Immunoreactive epitopic sites of these proteins which are similar to protein E epitope can create an immune stimulation on T cytotoxic and T helper cells and 6 of these 9 epitopic sites are also vaxiJen. These result imply that autoimmune cross-reaction is likely between the studied RPE proteins and SARS CoV-2 E protein. The structure of SARS CoV-2, its proteins and immunologic reactions against these proteins remain largely unknown. Understanding the structure of SARS CoV-2 proteins and demonstration of similarity with human proteins are crucial to predict an autoimmune response associated with immunity against host proteins and its clinical manifestations as well as possible adverse effects of vaccination.


Assuntos
Sequência de Aminoácidos , Doenças Autoimunes/virologia , Proteínas do Olho/química , Doenças Retinianas/virologia , Homologia de Sequência , Proteínas Virais/química , /epidemiologia , Biologia Computacional , /química , Infecções Oculares Virais/virologia , Humanos , Glicoproteínas de Membrana/química , Fosfoproteínas/química , Poliproteínas/química , Epitélio Pigmentado da Retina/química , Proteínas da Matriz Viral/química
12.
Nat Commun ; 12(1): 502, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479198

RESUMO

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.


Assuntos
/virologia , RNA Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , /genética , Membrana Celular/virologia , /genética , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Viral/genética , /genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
13.
Commun Biol ; 4(1): 129, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514825

RESUMO

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , /química , Glicoproteína da Espícula de Coronavírus/química , Adulto , /imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , /virologia , Convalescença , /metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Soros Imunes/química , Imunidade Humoral , Lentivirus/genética , Lentivirus/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , /patogenicidade , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Análise de Sobrevida
14.
J Basic Microbiol ; 61(3): 180-202, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33460172

RESUMO

The outbreak of a novel coronavirus associated with acute respiratory disease, called COVID-19, marked the introduction of the third spillover of an animal coronavirus (CoV) to humans in the last two decades. The genome analysis with various bioinformatics tools revealed that the causative pathogen (SARS-CoV-2) belongs to the subgenus Sarbecovirus of the genus Betacoronavirus, with highly similar genome as bat coronavirus and receptor-binding domain (RBD) of spike glycoprotein as Malayan pangolin coronavirus. Based on its genetic proximity, SARS-CoV-2 is likely to have originated from bat-derived CoV and transmitted to humans via an unknown intermediate mammalian host, probably Malayan pangolin. Further, spike protein S1/S2 cleavage site of SARS-CoV-2 has acquired polybasic furin cleavage site which is absent in bat and pangolin suggesting natural selection either in an animal host before zoonotic transfer or in humans following zoonotic transfer. In the current review, we recapitulate a preliminary opinion about the disease, origin and life cycle of SARS-CoV-2, roles of virus proteins in pathogenesis, commonalities, and differences between different corona viruses. Moreover, the crystal structures of SARS-CoV-2 proteins with unique characteristics differentiating it from other CoVs are discussed. Our review also provides comprehensive information on the molecular aspects of SARS-CoV-2 including secondary structures in the genome and protein-protein interactions which can be useful to understand the aggressive spread of the SARS-CoV-2. The mutations and the haplotypes reported in the SARS-CoV-2 genome are summarized to understand the virus evolution.


Assuntos
/virologia , /metabolismo , Animais , /química , /metabolismo , Genoma Viral , Humanos , Pandemias , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Replicação Viral
15.
BMC Complement Med Ther ; 21(1): 41, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478471

RESUMO

BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.


Assuntos
Alérgenos/química , Alérgenos/imunologia , /imunologia , Odorantes , Perfumes/química , /imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administração & dosagem , Alérgenos/uso terapêutico , Benzopiranos/química , Benzopiranos/metabolismo , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Cinamatos/química , Cinamatos/metabolismo , /metabolismo , /metabolismo , Diterpenos/química , Diterpenos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Perfumes/administração & dosagem , Perfumes/uso terapêutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
16.
Biophys J ; 120(6): 1085-1096, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33460600

RESUMO

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access.


Assuntos
Revisão por Pares , /química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Anticorpos Antivirais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Modelos Moleculares , Nucleocapsídeo/química , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Zinco/metabolismo
17.
Methods Mol Biol ; 2130: 185-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33284445

RESUMO

Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.


Assuntos
Ritmo Circadiano , Espectrometria de Massas/métodos , Fosfoproteínas/química , Proteômica/métodos , Animais , Camundongos , Fosfoproteínas/metabolismo , Proteoma/química , Proteoma/metabolismo
18.
Anal Chem ; 93(3): 1826-1833, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33370087

RESUMO

Collection of nasopharyngeal samples using swabs followed by the transfer of the virus into a solution and an RNA extraction step to perform reverse transcription polymerase chain reaction (PCR) is the primary method currently used for the diagnosis of COVID-19. However, the need for several reagents and steps and the high cost of PCR hinder its worldwide implementation to contain the outbreak. Here, we report a cotton-tipped electrochemical immunosensor for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus antigen. Unlike the reported approaches, we integrated the sample collection and detection tools into a single platform by coating screen-printed electrodes with absorbing cotton padding. The immunosensor was fabricated by immobilizing the virus nucleocapsid (N) protein on carbon nanofiber-modified screen-printed electrodes which were functionalized by diazonium electrografting. The detection of the virus antigen was achieved via swabbing followed by competitive assay using a fixed amount of N protein antibody in the solution. A square wave voltammetric technique was used for the detection. The limit of detection for our electrochemical biosensor was 0.8 pg/mL for SARS-CoV-2, indicating very good sensitivity for the sensor. The biosensor did not show significant cross-reactivity with other virus antigens such as influenza A and HCoV, indicating high selectivity of the method. Moreover, the biosensor was successfully applied for the detection of the virus antigen in spiked nasal samples showing excellent recovery percentages. Thus, our electrochemical immunosensor is a promising diagnostic tool for the direct rapid detection of the COVID-19 virus that requires no sample transfer or pretreatment.


Assuntos
/diagnóstico , Fibra de Algodão , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , /isolamento & purificação , Anticorpos Antivirais/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Carbono/química , /imunologia , Técnicas Eletroquímicas/instrumentação , Eletrodos , Gossypium/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Imunoensaio/instrumentação , Limite de Detecção , Nanofibras/química , Fosfoproteínas/química , Fosfoproteínas/imunologia , /imunologia
19.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333932

RESUMO

Uveal melanoma (UM) is the most common primary intraocular malignancy of the eye. It has a high metastatic potential and mainly spreads to the liver. Genetics play a vital role in tumor classification and prognostication of UM metastatic disease. One of the driver genes mutated in metastasized UM is subunit 1 of splicing factor 3b (SF3B1), a component of the spliceosome complex. Recurrent mutations in components of the spliceosome complex are observed in UM and other malignancies, suggesting an important role in tumorigenesis. SF3B1 is the most common mutated spliceosome gene and in UM it is associated with late-onset metastasis. This review summarizes the genetic and epigenetic insights of spliceosome mutations in UM. They form a distinct subgroup of UM and have similarities with other spliceosome mutated malignancies.


Assuntos
Melanoma/genética , Mutação , Fatores de Processamento de RNA/genética , Neoplasias Uveais/genética , Substituição de Aminoácidos , Éxons , Frequência do Gene , Humanos , Melanoma/metabolismo , Melanoma/mortalidade , Melanoma/patologia , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento de RNA , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos , Telômero/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/mortalidade , Neoplasias Uveais/patologia
20.
J Med Chem ; 63(24): 15773-15784, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33314931

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estabilidade de Medicamentos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Mutação , Domínios PDZ , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
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