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1.
Nat Commun ; 11(1): 4136, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811816

RESUMO

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/química , Nucleossomos/química , Nucleossomos/genética , Motivos de Aminoácidos/genética , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/ultraestrutura , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Dedos de Zinco/genética
2.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
3.
Nat Commun ; 11(1): 4171, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820176

RESUMO

Spiralia is a large, ancient and diverse clade of animals, with a conserved early developmental program but diverse larval and adult morphologies. One trait shared by many spiralians is the presence of ciliary bands used for locomotion and feeding. To learn more about spiralian-specific traits we have examined the expression of 20 genes with protein motifs that are strongly conserved within the Spiralia, but not detectable outside of it. Here, we show that two of these are specifically expressed in the main ciliary band of the mollusc Tritia (also known as Ilyanassa). Their expression patterns in representative species from five more spiralian phyla-the annelids, nemerteans, phoronids, brachiopods and rotifers-show that at least one of these, lophotrochin, has a conserved and specific role in particular ciliated structures, most consistently in ciliary bands. These results highlight the potential importance of lineage-specific genes or protein motifs for understanding traits shared across ancient lineages.


Assuntos
Motivos de Aminoácidos/genética , Cílios/genética , Invertebrados/genética , Proteínas/genética , Animais , Anelídeos/classificação , Anelídeos/genética , Anelídeos/fisiologia , Evolução Biológica , Cílios/fisiologia , Comportamento Alimentar/fisiologia , Perfilação da Expressão Gênica/métodos , Invertebrados/classificação , Invertebrados/fisiologia , Larva/genética , Larva/fisiologia , Locomoção/fisiologia , Moluscos/classificação , Moluscos/genética , Moluscos/fisiologia , Filogenia , Especificidade da Espécie
4.
BMC Bioinformatics ; 21(1): 340, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738892

RESUMO

BACKGROUND: Ribosome profiling has been widely used for studies of translation under a large variety of cellular and physiological contexts. Many of these studies have greatly benefitted from a series of data-mining tools designed for dissection of the translatome from different aspects. However, as the studies of translation advance quickly, the current toolbox still falls in short, and more specialized tools are in urgent need for deeper and more efficient mining of the important and new features of the translation landscapes. RESULTS: Here, we present RiboMiner, a bioinformatics toolset for mining of multi-dimensional features of the translatome with ribosome profiling data. RiboMiner performs extensive quality assessment of the data and integrates a spectrum of tools for various metagene analyses of the ribosome footprints and for detailed analyses of multiple features related to translation regulation. Visualizations of all the results are available. Many of these analyses have not been provided by previous methods. RiboMiner is highly flexible, as the pipeline could be easily adapted and customized for different scopes and targets of the studies. CONCLUSIONS: Applications of RiboMiner on two published datasets did not only reproduced the main results reported before, but also generated novel insights into the translation regulation processes. Therefore, being complementary to the current tools, RiboMiner could be a valuable resource for dissections of the translation landscapes and the translation regulations by mining the ribosome profiling data more comprehensively and with higher resolution. RiboMiner is freely available at https://github.com/xryanglab/RiboMiner and https://pypi.org/project/RiboMiner .


Assuntos
Biologia Computacional/métodos , Biossíntese de Proteínas , Ribossomos/metabolismo , Software , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/genética , Códon/genética , Análise de Dados , Mineração de Dados
5.
Int J Antimicrob Agents ; 56(3): 106119, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32738306

RESUMO

Coronavirus disease 2019 (COVID-19) is a highly transmissible viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical trials have reported improved outcomes resulting from an effective reduction or absence of viral load when patients were treated with chloroquine (CQ) or hydroxychloroquine (HCQ). In addition, the effects of these drugs were improved by simultaneous administration of azithromycin (AZM). The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein binds to the cell surface angiotensin-converting enzyme 2 (ACE2) receptor, allowing virus entry and replication in host cells. The viral main protease (Mpro) and host cathepsin L (CTSL) are among the proteolytic systems involved in SARS-CoV-2 S protein activation. Hence, molecular docking studies were performed to test the binding performance of these three drugs against four targets. The findings showed AZM affinity scores (ΔG) with strong interactions with ACE2, CTSL, Mpro and RBD. CQ affinity scores showed three low-energy results (less negative) with ACE2, CTSL and RBD, and a firm bond score with Mpro. For HCQ, two results (ACE2 and Mpro) were firmly bound to the receptors, however CTSL and RBD showed low interaction energies. The differences in better interactions and affinity between HCQ and CQ with ACE2 and Mpro were probably due to structural differences between the drugs. On other hand, AZM not only showed more negative (better) values in affinity, but also in the number of interactions in all targets. Nevertheless, further studies are needed to investigate the antiviral properties of these drugs against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Azitromicina/química , Betacoronavirus/química , Catepsina L/química , Cloroquina/química , Cisteína Endopeptidases/química , Hidroxicloroquina/química , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Antivirais/química , Azitromicina/farmacologia , Betacoronavirus/metabolismo , Sítios de Ligação , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Hidroxicloroquina/farmacologia , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Ligação Viral/efeitos dos fármacos
6.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769995

RESUMO

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Tripsina/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-32669265

RESUMO

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Inibidores de Proteases/química , Pirrolidinas/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Chlorocebus aethiops , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Expressão Gênica , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirrolidinas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Nat Commun ; 11(1): 3748, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719311

RESUMO

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a ß-hairpin loop, and interconnected along the tail by the splayed ß-hairpins. By contrast, we show that the ß-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.


Assuntos
Bacteriófagos/fisiologia , Flagelos/fisiologia , Motivos de Aminoácidos , Bacteriófagos/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , DNA/genética , DNA Viral/genética , Flagelos/ultraestrutura , Herpesviridae/ultraestrutura , Multimerização Proteica , Estrutura Secundária de Proteína , Vírion/ultraestrutura , Vitrificação
9.
Nat Commun ; 11(1): 3383, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636391

RESUMO

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.


Assuntos
Lesão Renal Aguda/genética , Elementos Facilitadores Genéticos , Túbulos Renais/citologia , Lesão Renal Aguda/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células , Epigênese Genética , Fibrose , Fator 4 Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Nucleares , Receptores de Glucocorticoides/metabolismo , Elementos Reguladores de Transcrição , Traumatismo por Reperfusão/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Genética
10.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
11.
Nat Commun ; 11(1): 3583, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681005

RESUMO

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
12.
PLoS Negl Trop Dis ; 14(7): e0008436, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639997

RESUMO

Nodding syndrome (NS) is a devastating and enigmatic childhood epilepsy. NS is accompanied by multiple neurological impairments and neuroinflammation, and associated with the parasite Onchocerca volvulus (Ov) and other environmental factors. Moreover, NS seems to be an 'Autoimmune Epilepsy' since: 1. ~50% of NS patients have neurotoxic cross-reactive Ov/Leimodin-I autoimmune antibodies. 2. Our recently published findings: Most (~86%) of NS patients have glutamate-receptor AMPA-GluR3B peptide autoimmune antibodies that bind, induce Reactive Oxygen Species, and kill both neural cells and T cells. Furthermore, NS patient's IgG induce seizures, brain multiple damage alike occurring in brains of NS patients, and elevation of T cells and activated microglia and astrocytes, in brains of normal mice. Human Leukocyte antigen (HLA) class I and II molecules are critical for initiating effective beneficial immunity against foreign microorganisms and contributing to proper brain function, but also predispose to detrimental autoimmunity against self-peptides. We analyzed seven HLA loci, either by next-generation-sequencing or Sequence-Specific-Oligonucleotide-Probe, in 48 NS patients and 51 healthy controls from South Sudan. We discovered that NS associates significantly with both protective HLA haplotype: HLA-B*42:01, C*17:01, DRB1*03:02, DQB1*04:02 and DQA1*04:01, and susceptible motif: Ala24, Glu63 and Phe67, in the HLA-B peptide-binding groove. These amino acids create a hydrophobic and sterically closed peptide-binding HLA pocket, favoring proline residue. Our findings suggest that immunogenetic fingerprints in HLA peptide-binding grooves tentatively associate with protection or susceptibility to NS. Accordingly, different HLA molecules may explain why under similar environmental factors, only some children, within the same families, tribes and districts, develop NS, while others do not.


Assuntos
Antígenos HLA/química , Antígenos HLA/imunologia , Síndrome do Cabeceio/imunologia , Adolescente , Adulto , Motivos de Aminoácidos , Autoanticorpos/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Suscetibilidade a Doenças , Feminino , Antígenos HLA/genética , Humanos , Masculino , Síndrome do Cabeceio/genética , Síndrome do Cabeceio/prevenção & controle , Receptores de AMPA/genética , Receptores de AMPA/imunologia , Sudão do Sul , Adulto Jovem
13.
J Transl Med ; 18(1): 281, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650788

RESUMO

BACKGROUND: The recent outbreak by SARS-CoV-2 has generated a chaos in global health and economy and claimed/infected a large number of lives. Closely resembling with SARS CoV, the present strain has manifested exceptionally higher degree of spreadability, virulence and stability possibly due to some unidentified mutations. The viral spike glycoprotein is very likely to interact with host Angiotensin-Converting Enzyme 2 (ACE2) and transmits its genetic materials and hijacks host machinery with extreme fidelity for self propagation. Few attempts have been made to develop a suitable vaccine or ACE2 blocker or virus-receptor inhibitor within this short period of time. METHODS: Here, attempt was taken to develop some therapeutic and vaccination strategies with a comparison of spike glycoproteins among SARS-CoV, MERS-CoV and the SARS-CoV-2. We verified their structure quality (SWISS-MODEL, Phyre2, and Pymol) topology (ProFunc), motifs (MEME Suite, GLAM2Scan), gene ontology based conserved domain (InterPro database) and screened several epitopes (SVMTrip) of SARS CoV-2 based on their energetics, IC50 and antigenicity with regard to their possible glycosylation and MHC/paratope binding (Vaxigen v2.0, HawkDock, ZDOCK Server) effects. RESULTS: We screened here few pairs of spike protein epitopic regions and selected their energetic, Inhibitory Concentration50 (IC50), MHC II reactivity and found some of those to be very good target for vaccination. A possible role of glycosylation on epitopic region showed profound effects on epitopic recognition. CONCLUSION: The present work might be helpful for the urgent development of a suitable vaccination regimen against SARS CoV-2.


Assuntos
Betacoronavirus/imunologia , Biologia Computacional/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Infecções por Coronavirus/prevenção & controle , Glicosilação , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Concentração Inibidora 50 , Anotação de Sequência Molecular , Pandemias , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/química
14.
Nat Commun ; 11(1): 3618, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681106

RESUMO

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human-infecting coronaviruses is still lacking. We used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post-fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Motivos de Aminoácidos , Coronavirus/química , Coronavirus/classificação , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Humanos , Fusão de Membrana , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Multimerização Proteica , Internalização do Vírus
15.
Comput Biol Med ; 122: 103849, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658736

RESUMO

SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be "hiding" sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.


Assuntos
Betacoronavirus/química , Biologia Computacional , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Conformação Molecular , Ácido N-Acetilneuramínico/química , Pandemias , Polissacarídeos/química , Vírus da SARS , Triptofano/química
17.
Nucleic Acids Res ; 48(13): 7532-7544, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32501503

RESUMO

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.


Assuntos
Acetiltransferases/química , Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Aminoacilação de RNA de Transferência , Acetiltransferases/genética , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Especificidade por Substrato
18.
Nat Commun ; 11(1): 3219, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591542

RESUMO

The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. Most human RPTPs possess an extracellular receptor domain and tandem intracellular phosphatase domains: comprising an active membrane proximal (D1) domain and an inactive distal (D2) pseudophosphatase domain. Here we demonstrate that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal "backdoor" cysteine, a process thought to reversibly inactivate related phosphatases. Importantly, despite the absence of catalytic activity, PTPRU binds substrates of related phosphatases strongly suggesting that this pseudophosphatase functions in tyrosine phosphorylation by competing with active phosphatases for the binding of substrates.


Assuntos
Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Linhagem Celular , Dissulfetos/metabolismo , Estabilidade Enzimática , Humanos , Modelos Moleculares , Oxirredução , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Especificidade por Substrato
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