Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 272.887
Filtrar
1.
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019591

RESUMO

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.


Assuntos
Imunidade Inata , Proteínas de Transporte da Membrana Mitocondrial/química , Animais , Betacoronavirus/genética , Sítios de Ligação , Humanos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Transporte Proteico
2.
Sci Rep ; 10(1): 16471, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020502

RESUMO

SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.


Assuntos
Peptidil Dipeptidase A/química , Filogenia , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Humanos , Mamíferos , Simulação de Acoplamento Molecular , Mutação , Peptidil Dipeptidase A/classificação , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Opt Lett ; 45(19): 5428-5431, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33001920

RESUMO

We show that waveguide sensors can enable a quantitative characterization of coronavirus spike glycoprotein-host-receptor binding-the process whereby coronaviruses enter human cells, causing disease. We demonstrate that such sensors can help quantify and eventually understand kinetic and thermodynamic properties of viruses that control their affinity to targeted cells, which is known to significantly vary in the course of virus evolution, e.g., from SARS-CoV to SARS-CoV-2, making the development of virus-specific drugs and vaccine difficult. With the binding rate constants and thermodynamic parameters as suggested by the latest SARS-CoV-2 research, optical sensors of SARS-CoV-2 spike protein-receptor binding may be within sight.


Assuntos
Betacoronavirus , Técnicas Biossensoriais , Infecções por Coronavirus , Óptica e Fotônica/instrumentação , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica/fisiologia
4.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007253

RESUMO

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Assuntos
Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Animais , Catálise , Células HeLa , Humanos , Íntrons/genética , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de Tempo
5.
Mol Cell ; 80(1): 156-163.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007255

RESUMO

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
6.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024075
7.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
8.
Nat Commun ; 11(1): 4947, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009392

RESUMO

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Pseudomonas syringae/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Secreção Bacterianos , Sítios de Ligação , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Multimerização Proteica , Pseudomonas syringae/patogenicidade , Reprodutibilidade dos Testes , Técnica de Seleção de Aptâmeros , Virulência
9.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
10.
Sci Adv ; 6(35): eaba7910, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32923629

RESUMO

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Cinamatos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Influenza Humana/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , ortoaminobenzoatos/farmacologia , Células A549 , Animais , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cães , Células HEK293 , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/patologia
11.
Science ; 369(6508): 1166-1167, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32883850
12.
Nat Commun ; 11(1): 4420, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887876

RESUMO

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Betacoronavirus/efeitos dos fármacos , Camelídeos Americanos/imunologia , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/virologia , Ligação Proteica , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
13.
Molecules ; 25(18)2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927621

RESUMO

Mass spectrometry and some other biophysical methods, have made substantial contributions to the studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins interactions. The most interesting feature of SARS-CoV-2 seems to be the structure of its spike (S) protein and its interaction with the human cell receptor. Mass spectrometry of spike S protein revealed how the glycoforms are distributed across the S protein surface. X-ray crystallography and cryo-electron microscopy made huge impact on the studies on the S protein and ACE2 receptor protein interaction, by elucidating the three-dimensional structures of these proteins and their conformational changes. The findings of the most recent studies in the scope of SARS-CoV-2-Human protein-protein interactions are described here.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/epidemiologia , Receptores Virais/química , Síndrome Respiratória Aguda Grave/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/patogenicidade , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
Sci Data ; 7(1): 309, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938937

RESUMO

Emergence of coronaviruses poses a threat to global health and economy. The current outbreak of SARS-CoV-2 has infected more than 28,000,000 people and killed more than 915,000. To date, there is no treatment for coronavirus infections, making the development of therapies to prevent future epidemics of paramount importance. To this end, we collected information regarding naturally-occurring variants of the Angiotensin-converting enzyme 2 (ACE2), an epithelial receptor that both SARS-CoV and SARS-CoV-2 use to enter the host cells. We built 242 structural models of variants of human ACE2 bound to the receptor binding domain (RBD) of the SARS-CoV-2 surface spike glycoprotein (S protein) and refined their interfaces with HADDOCK. Our dataset includes 140 variants of human ACE2 representing missense mutations found in genome-wide studies, 39 mutants with reported effects on the recognition of the RBD, and 63 predictions after computational alanine scanning mutagenesis of ACE2-RBD interface residues. This dataset will help accelerate the design of therapeutics against SARS-CoV-2, as well as contribute to prevention of possible future coronaviruses outbreaks.


Assuntos
Desenho de Fármacos , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Betacoronavirus , Sítios de Ligação , Infecções por Coronavirus , Humanos , Modelos Moleculares , Pandemias , Pneumonia Viral , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Virais/química
15.
Biomolecules ; 10(9)2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32967116

RESUMO

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus , Sítios de Ligação , Cloroquina/química , Cloroquina/farmacologia , Infecções por Coronavirus/prevenção & controle , Reposicionamento de Medicamentos , Eflornitina/química , Eflornitina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ivermectina/química , Ivermectina/farmacologia , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral/prevenção & controle , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Receptores da Glicina/química , Receptores da Glicina/efeitos dos fármacos , Saposinas/química , Saposinas/efeitos dos fármacos , Sofosbuvir/química , Sofosbuvir/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Relação Estrutura-Atividade
16.
Nat Commun ; 11(1): 4578, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929090

RESUMO

Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of ß-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.


Assuntos
Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Estereoisomerismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Talidomida/química , Talidomida/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
Signal Transduct Target Ther ; 5(1): 212, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963228

RESUMO

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Síndrome Respiratória Aguda Grave/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/imunologia , Vírion/ultraestrutura
18.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
19.
Int J Antimicrob Agents ; 56(2): 106020, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32862840

RESUMO

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.


Assuntos
Azitromicina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Gangliosídeo G(M1)/metabolismo , Hidroxicloroquina/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Alinhamento de Sequência
20.
J Toxicol Sci ; 45(9): 569-579, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879256

RESUMO

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.


Assuntos
Indóis/efeitos adversos , Macrófagos/imunologia , Macrófagos/metabolismo , Oxirredução/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células HL-60 , Humanos , Indicã/metabolismo , Macrófagos/efeitos dos fármacos , Ligação Proteica , Albumina Sérica/metabolismo , Triptofano/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA