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1.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
2.
BMC Bioinformatics ; 21(Suppl 8): 262, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938371

RESUMO

BACKGROUND: Properly scoring protein-protein docking models to single out the correct ones is an open challenge, also object of assessment in CAPRI (Critical Assessment of PRedicted Interactions), a community-wide blind docking experiment. We introduced in the field CONSRANK (CONSensus RANKing), the first pure consensus method. Also available as a web server, CONSRANK ranks docking models in an ensemble based on their ability to match the most frequent inter-residue contacts in it. We have been blindly testing CONSRANK in all the latest CAPRI rounds, where we showed it to perform competitively with the state-of-the-art energy and knowledge-based scoring functions. More recently, we developed Clust-CONSRANK, an algorithm introducing a contact-based clustering of the models as a preliminary step of the CONSRANK scoring process. In the latest CASP13-CAPRI joint experiment, we participated as scorers with a novel pipeline, combining both our scoring tools, CONSRANK and Clust-CONSRANK, with our interface analysis tool COCOMAPS. Selection of the 10 models for submission was guided by the strength of the emerging consensus, and their final ranking was assisted by results of the interface analysis. RESULTS: As a result of the above approach, we were by far the first scorer in the CASP13-CAPRI top-1 ranking, having high/medium quality models ranked at the top-1 position for the majority of targets (11 out of the total 19). We were also the first scorer in the top-10 ranking, on a par with another group, and the second scorer in the top-5 ranking. Further, we topped the ranking relative to the prediction of binding interfaces, among all the scorers and predictors. Using the CASP13-CAPRI targets as case studies, we illustrate here in detail the approach we adopted. CONCLUSIONS: Introducing some flexibility in the final model selection and ranking, as well as differentiating the adopted scoring approach depending on the targets were the key assets for our highly successful performance, as compared to previous CAPRI rounds. The approach we propose is entirely based on methods made available to the community and could thus be reproduced by any user.


Assuntos
Biologia Computacional/métodos , Ligação Proteica/genética , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Algoritmos , Humanos , Conformação Proteica
3.
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
4.
Nat Commun ; 11(1): 4414, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887877

RESUMO

CD4+ helper T cells contribute important functions to the immune response during pathogen infection and tumor formation by recognizing antigenic peptides presented by class II major histocompatibility complexes (MHC-II). While many computational algorithms for predicting peptide binding to MHC-II proteins have been reported, their performance varies greatly. Here we present a yeast-display-based platform that allows the identification of over an order of magnitude more unique MHC-II binders than comparable approaches. These peptides contain previously identified motifs, but also reveal new motifs that are validated by in vitro binding assays. Training of prediction algorithms with yeast-display library data improves the prediction of peptide-binding affinity and the identification of pathogen-associated and tumor-associated peptides. In summary, our yeast-display-based platform yields high-quality MHC-II-binding peptide datasets that can be used to improve the accuracy of MHC-II binding prediction algorithms, and potentially enhance our understanding of CD4+ T cell recognition.


Assuntos
Epitopos de Linfócito T/genética , Oligopeptídeos , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Técnicas de Visualização da Superfície Celular , Bases de Dados de Proteínas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo
5.
ACS Nano ; 14(8): 10616-10623, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32806067

RESUMO

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Sítios de Ligação/genética , Desenho de Fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
6.
Nat Commun ; 11(1): 4267, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848148

RESUMO

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Zigoto/metabolismo , Animais , Sítios de Ligação/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Feminino , Genoma Humano , Proteínas de Homeodomínio/metabolismo , Humanos , Cinética , Camundongos , Regiões Promotoras Genéticas , Estudo de Prova de Conceito , Ligação Proteica/genética , Especificidade da Espécie
7.
PLoS One ; 15(8): e0237559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780783

RESUMO

BACKGROUND: The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5' and 3' untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. METHODS: To have an idea of the global changes in 5' and 3'-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by 'RBPmap' and 'RNA22 v2' respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. RESULTS: We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5'-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3'UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Betacoronavirus/genética , Infecções por Coronavirus/metabolismo , Genoma Viral/genética , Instabilidade Genômica/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/metabolismo , Pneumonia Viral/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Ligação Proteica/genética
8.
Med Sci (Paris) ; 36(8-9): 717-724, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32821048

RESUMO

Ribosome display is a powerful method for selection and molecular evolution of proteins and peptides from large libraries. Displayed proteins are recovered from target molecules in multiple rounds of selection in order to enrich specific binders with the desired properties. Nowadays, ribosome display has become one of the most widely-used display technologies thanks to its advantages over cell-display as phage display. Ribosome display is an in vitro method, in which a stable ternary complex is formed between the mRNA, the ribosome and the nascent protein. A selection cycle can be performed in a few days and bacterial transformation is not necessary. Ribosome display has been used to screen and select peptides, proteins or molecular scaffolds in order to increase their affinity, specificity, catalytic activity or stability. In this review, ribosome display systems and their applications in selection and evolution of proteins are described.


Assuntos
Sistema Livre de Células/metabolismo , Evolução Molecular Direcionada/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Sítios de Ligação/genética , Sistema Livre de Células/química , Perfilação da Expressão Gênica/tendências , Humanos , Ligação Proteica/genética , Proteínas Ribossômicas/metabolismo
9.
Nucleic Acids Res ; 48(15): 8724-8739, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32735645

RESUMO

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Motivated by our previous finding that intron retention (IR) could lead to transcript instability, in this study, we performed BruChase-Seq to experimentally monitor the expression dynamics of nascent transcripts in resting and activated CD4+ T cells. Computational modeling was then applied to quantify the stability of spliced and intron-retained transcripts on a genome-wide scale. Beyond substantiating that intron-retained transcripts were considerably less stable than spliced transcripts, we found a global stabilization of spliced mRNAs upon T cell activation, although the stability of intron-retained transcripts remained relatively constant. In addition, we identified that La-related protein 4 (LARP4), an RNA-binding protein (RBP) known to enhance mRNA stability, was involved in T cell activation-dependent mRNA stabilization. Knocking out Larp4 in mice destabilized Nfκb1 mRNAs and reduced secretion of interleukin-2 (IL2) and interferon-gamma (IFNγ), two factors critical for T cell proliferation and function. We propose that coordination between splicing regulation and mRNA stability may provide a novel paradigm to control spatiotemporal gene expression during T cell activation.


Assuntos
Interferon gama/genética , Interleucina-2/genética , Proteínas/genética , Estabilidade de RNA/genética , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Humanos , Íntrons/genética , Ativação Linfocitária/genética , Camundongos , NF-kappa B/genética , Ligação Proteica/genética , RNA Mensageiro/genética , Linfócitos T/metabolismo
10.
Nucleic Acids Res ; 48(15): 8529-8544, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738045

RESUMO

Myocyte enhancer factor-2B (MEF2B) has the unique capability of binding to its DNA target sites with a degenerate motif, while still functioning as a gene-specific transcriptional regulator. Identifying its DNA targets is crucial given regulatory roles exerted by members of the MEF2 family and MEF2B's involvement in B-cell lymphoma. Analyzing structural data and SELEX-seq experimental results, we deduced the DNA sequence and shape determinants of MEF2B target sites on a high-throughput basis in vitro for wild-type and mutant proteins. Quantitative modeling of MEF2B binding affinities and computational simulations exposed the DNA readout mechanisms of MEF2B. The resulting binding signature of MEF2B revealed distinct intricacies of DNA recognition compared to other transcription factors. MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif, where A-tract polarity dictates nuances of binding. The predominant role of shape readout at the center of the core motif, with most contacts formed in the minor groove, differs from previously observed protein-DNA readout modes. MEF2B, therefore, represents a unique protein for studies of the role of DNA shape in achieving binding specificity. MEF2B-DNA recognition mechanisms are likely representative for other members of the MEF2 family.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/ultraestrutura , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/ultraestrutura , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica/genética
11.
Nucleic Acids Res ; 48(15): 8562-8575, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32749456

RESUMO

Eukaryotic cellular mRNAs possess a 5' cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.


Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/genética , Biossíntese de Proteínas , Sequência de Aminoácidos/genética , Animais , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4F em Eucariotos/química , Células HEK293 , Humanos , Camundongos , Ligação Proteica/genética , Análogos de Capuz de RNA/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , Ribossomos/genética
12.
PLoS Genet ; 16(8): e1008955, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776921

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by excess lipid accumulation in the liver without significant consumption of alcohol. The transmembrane 6 superfamily member 2 (TM6SF2) E167K missense variant strongly associates with NAFLD in humans. The E167K mutation destabilizes TM6SF2, resulting in hepatic lipid accumulation and low serum lipid levels. However, the molecular mechanism by which TM6SF2 regulates lipid metabolism remains unclear. By using tandem affinity purification in combination with mass spectrometry, we found that apolipoprotein B (APOB), ER lipid raft protein (ERLIN) 1 and 2 were TM6SF2-interacting proteins. ERLINs and TM6SF2 mutually bound and stabilized each other. TM6SF2 bound and stabilized APOB via two luminal loops. ERLINs did not interact with APOB directly but still increased APOB stability through stabilizing TM6SF2. This APOB stabilization was hampered by the E167K mutation that reduced the protein expression of TM6SF2. In mice, knockout of Tm6sf2 and knockdown of Tm6sf2 or Erlins decreased hepatic APOB protein level, causing lipid accumulation in the liver and lowering lipid levels in the serum. We conclude that defective APOB stabilization, as a result of ERLINs or TM6SF2 deficiency or E167K mutation, is a key factor contributing to NAFLD.


Assuntos
Apolipoproteína B-100/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Colesterol/genética , Colesterol/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Imunoprecipitação , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Lipídeos/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/genética , Transfecção
13.
Hypertension ; 76(5): 1339-1349, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32851855

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 originated from Wuhan, China, in December 2019 and rapidly spread to other areas worldwide. Since then, coronavirus disease 2019 (COVID-19) has reached pandemic proportions with >570 000 deaths globally by mid-July 2020. The magnitude of the outbreak and the potentially severe clinical course of COVID-19 has led to a burst of scientific research on this novel coronavirus and its host receptor ACE (angiotensin-converting enzyme)-2. ACE2 is a homolog of the ACE that acts on several substrates in the renin-Ang (angiotensin) system. With unprecedented speed, scientific research has solved the structure of SARS-CoV-2 and imaged its binding with the ACE2 receptor. In SARS-CoV-2 infection, the viral S (spike) protein receptor-binding domain binds to ACE2 to enter the host cell. ACE2 expression in the lungs is relatively low, but it is present in type II pneumocytes-a cell type also endowed with TMPRSS2 (transmembrane protease serine 2). This protease is critical for priming the SARS-CoV-2 S protein to complex with ACE2 and enter the cells. Herein, we review the current understanding of the interaction of SARS-CoV-2 with ACE2 as it has rapidly unfolded over the last months. While it should not be assumed that we have a complete picture of SARS-CoV-2 mechanism of infection and its interaction with ACE2, much has been learned with clear therapeutic implications. Potential therapies aimed at intercepting SARS-CoV-2 from reaching the full-length membrane-bound ACE2 receptor using soluble ACE2 protein and other potential approaches are briefly discussed as well.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Betacoronavirus/metabolismo , Infecções por Coronavirus/epidemiologia , Pandemias/estatística & dados numéricos , Peptidil Dipeptidase A/genética , Pneumonia Viral/epidemiologia , Ligação Proteica/genética , China , Infecções por Coronavirus/metabolismo , Surtos de Doenças/estatística & dados numéricos , Feminino , Humanos , Masculino , Pandemias/prevenção & controle , Pneumonia Viral/metabolismo , RNA Viral/genética
14.
PLoS Genet ; 16(6): e1008865, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603360

RESUMO

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.


Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Calcineurina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição Forkhead/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Grupo de Alta Mobilidade/genética , Peptidilprolil Isomerase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética
15.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(1): 71-75, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32621420

RESUMO

OBJECTIVE: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (NaV1.2 IQ). METHODS: The cDNA of NaV1.2 IQ was constructed by PCR technique, CaM mutants CaM12, CaM34 and CaM1234 were constructed with QuickchangeTM site-directed mutagenesis kit (QIAGEN). The binding of NaV1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. RESULTS: NaV1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and NaV1.2 IQ at [Ca2+]-free was greater than that at 100 nmol/L [Ca2+] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to NaV1.2 IQ was greater than that of its mutant, and the binding affinity of CaM1234 to NaV1.2 IQ was the weakest among the three mutants (P < 0.05). CONCLUSIONS: The binding ability of CaM and CaM mutants to NaV1.2 IQ is Ca2+-dependent. This study has revealed a new mechanism of NaV1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.


Assuntos
Calmodulina , Canal de Sódio Disparado por Voltagem NAV1.2 , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Ligação Proteica/genética
16.
J Mol Biol ; 432(19): 5212-5226, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32710986

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is a major concern in coronavirus disease 2019 (COVID-19) prevention and economic reopening. However, rigorous determination of SARS-CoV-2 infectivity is very difficult owing to its continuous evolution with over 10,000 single nucleotide polymorphisms (SNP) variants in many subtypes. We employ an algebraic topology-based machine learning model to quantitatively evaluate the binding free energy changes of SARS-CoV-2 spike glycoprotein (S protein) and host angiotensin-converting enzyme 2 receptor following mutations. We reveal that the SARS-CoV-2 virus becomes more infectious. Three out of six SARS-CoV-2 subtypes have become slightly more infectious, while the other three subtypes have significantly strengthened their infectivity. We also find that SARS-CoV-2 is slightly more infectious than SARS-CoV according to computed S protein-angiotensin-converting enzyme 2 binding free energy changes. Based on a systematic evaluation of all possible 3686 future mutations on the S protein receptor-binding domain, we show that most likely future mutations will make SARS-CoV-2 more infectious. Combining sequence alignment, probability analysis, and binding free energy calculation, we predict that a few residues on the receptor-binding motif, i.e., 452, 489, 500, 501, and 505, have high chances to mutate into significantly more infectious COVID-19 strains.


Assuntos
Betacoronavirus/genética , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Evolução Molecular , Mutação , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Betacoronavirus/classificação , Análise por Conglomerados , Análise Mutacional de DNA , Genótipo , Mapeamento Geográfico , Humanos , Aprendizado de Máquina , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Probabilidade , Ligação Proteica/genética , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/genética , Vírus da SARS/metabolismo , Vírus da SARS/patogenicidade , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Termodinâmica
17.
Nature ; 585(7823): 85-90, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32699409

RESUMO

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1-3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4-6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.


Assuntos
Glipicanas/química , Glipicanas/metabolismo , Lipídeos , Transdução de Sinais , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Feminino , Glipicanas/classificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Masculino , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Domínios Proteicos , Transporte Proteico , Solubilidade , Proteína Wnt1/química , Proteína Wnt1/metabolismo
18.
Nucleic Acids Res ; 48(15): 8755-8766, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621606

RESUMO

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr-GPSGCC and SBDSco-GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA-protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.


Assuntos
Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Enxofre/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , DNA/química , Enzimas de Restrição do DNA/química , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Ligação de Hidrogênio , Ligação Proteica/genética , Domínios Proteicos/genética , Streptomyces/enzimologia
19.
Nucleic Acids Res ; 48(15): 8332-8348, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633757

RESUMO

Negative cofactor 2 (NC2), including two subunits NC2α and NC2ß, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2ß interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2ß is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.


Assuntos
Catalase/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcrição Genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Genes MHC da Classe II/genética , Histonas/genética , Neurospora crassa/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Fosfoproteínas/ultraestrutura , Ligação Proteica/genética , Fatores de Transcrição/ultraestrutura , Ativação Transcricional/genética
20.
Proc Natl Acad Sci U S A ; 117(31): 18701-18710, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690679

RESUMO

Yin Yang 1 (YY1) is a DNA-binding transcription factor that either activates or represses gene expression. YY1 has previously been implicated in the transcriptional silencing of many retroviruses by binding to DNA sequences in the U3 region of the viral long terminal repeat (LTR). We here show that YY1 overexpression leads to profound activation, rather than repression, of human T lymphotropic virus type 1 (HTLV-1) expression, while YY1 down-regulation reduces HTLV-1 expression. The YY1 responsive element mapped not to YY1 DNA-binding sites in the HTLV-1 LTR but to the R region. The HTLV-1 R sequence alone is sufficient to provide YY1 responsiveness to a nonresponsive promoter, but only in the sense orientation and only when included as part of the mRNA. YY1 binds to the R region of HTLV-1 RNA in vitro and in vivo, leading to increased transcription initiation and elongation. The findings indicate that YY1 is a potent transactivator of HTLV-1 gene expression acting via binding viral RNA, rather than DNA.


Assuntos
Regulação Viral da Expressão Gênica/genética , Vírus Linfotrópico T Tipo 1 Humano , RNA/metabolismo , Sequências Repetidas Terminais/genética , Fator de Transcrição YY1 , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Ligação Proteica/genética , RNA/genética , Ativação Transcricional/genética , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
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