RESUMO
Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.
Assuntos
Cromatina , Desenvolvimento Embrionário , Peixe-Zebra , Animais , Cromatina/genética , Cromatina/metabolismo , Gema de Ovo/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Análise de Célula Única , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We present a major update of the HOCOMOCO collection that provides DNA binding specificity patterns of 949 human transcription factors and 720 mouse orthologs. To make this release, we performed motif discovery in peak sets that originated from 14 183 ChIP-Seq experiments and reads from 2554 HT-SELEX experiments yielding more than 400 thousand candidate motifs. The candidate motifs were annotated according to their similarity to known motifs and the hierarchy of DNA-binding domains of the respective transcription factors. Next, the motifs underwent human expert curation to stratify distinct motif subtypes and remove non-informative patterns and common artifacts. Finally, the curated subset of 100 thousand motifs was supplied to the automated benchmarking to select the best-performing motifs for each transcription factor. The resulting HOCOMOCO v12 core collection contains 1443 verified position weight matrices, including distinct subtypes of DNA binding motifs for particular transcription factors. In addition to the core collection, HOCOMOCO v12 provides motif sets optimized for the recognition of binding sites in vivo and in vitro, and for annotation of regulatory sequence variants. HOCOMOCO is available at https://hocomoco12.autosome.org and https://hocomoco.autosome.org.
Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição , Animais , Humanos , Camundongos , Sítios de Ligação/genética , Motivos de Nucleotídeos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Internet , Domínios e Motivos de Interação entre Proteínas/genéticaRESUMO
Mutations, especially those at the protein-protein interaction (PPI) interface, have been associated with various diseases. Meanwhile, though de novo mutations (DNMs) have been proven important in neuropsychiatric disorders, such as developmental delay (DD), the relationship between PPI interface DNMs and DD has not been well studied. Here we curated developmental delay DNM datasets from the PsyMuKB database and showed that DD patients showed a higher rate and deleteriousness in DNM missense on the PPI interface than sibling control. Next, we identified 302 DD-related PsychiPPIs, defined as PPIs harboring a statistically significant number of DNM missenses at their interface, and 42 DD candidate genes from PsychiPPI. We observed that PsychiPPIs preferentially affected the human protein interactome network hub proteins. When analyzing DD candidate genes using gene ontology and gene spatio-expression, we found that PsychiPPI genes carrying PPI interface mutations, such as FGFR3 and ALOX5, were enriched in development-related pathways and the development of the neocortex, and cerebellar cortex, suggesting their potential involvement in the etiology of DD. Our results demonstrated that DD patients carried an excess burden of PPI-truncating DNM, which could be used to efficiently search for disease-related genes and mutations in large-scale sequencing studies. In conclusion, our comprehensive study indicated the significant role of PPI interface DNMs in developmental delay pathogenicity.
Assuntos
Mutação , Domínios e Motivos de Interação entre Proteínas , Humanos , Domínios e Motivos de Interação entre Proteínas/genéticaRESUMO
The omicron variant of SARS-CoV-2 has been spreading rapidly across the globe. The virus-surface spike protein plays a critical role in the cell entry and immune evasion of SARS-CoV-2. Here we determined the 3.0 Å cryo-EM structure of the omicron spike protein ectodomain. In contrast to the original strain of SARS-CoV-2 where the receptor-binding domain (RBD) of the spike protein takes a mixture of open ("standing up") and closed ("lying down") conformations, the omicron spike molecules are predominantly in the open conformation, with one upright RBD ready for receptor binding. The open conformation of the omicron spike is stabilized by enhanced inter-domain and inter-subunit packing, which involves new mutations in the omicron strain. Moreover, the omicron spike has undergone extensive mutations in RBD regions where known neutralizing antibodies target, allowing the omicron variant to escape immune surveillance aimed at the original viral strain. The stable open conformation of the omicron spike sheds light on the cell entry and immune evasion mechanisms of the omicron variant.
Assuntos
COVID-19/virologia , SARS-CoV-2/química , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Microscopia Crioeletrônica , Humanos , Evasão da Resposta Imune/genética , Modelos Moleculares , Mutação , Pandemias , Conformação Proteica , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Internalização do VírusRESUMO
Plakin repeat domains (PRDs) are globular modules that mediate the interaction of plakin proteins with the intermediate filament (IF) cytoskeleton. These associations are vital for maintaining tissue integrity in cardiac muscle and epithelial tissues. PRDs are subject to mutations that give rise to cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy, characterised by ventricular arrhythmias and associated with an increased risk of sudden heart failure, and skin blistering diseases. Herein, we have examined the functional and structural effects of 12 disease-linked missense mutations, identified from the human gene mutation database, on the PRDs of the desmosomal protein desmoplakin. Five mutations (G2056R and E2193K in PRD-A, G2338R and G2375R in PRD-B and G2647D in PRD-C) rendered their respective PRD proteins either fully or partially insoluble following expression in bacterial cells. Each of the residues affected are conserved across plakin family members, inferring a crucial role in maintaining the structural integrity of the PRD. In transfected HeLa cells, the mutation G2375R adversely affected the targeting of a desmoplakin C-terminal construct containing all three PRDs to vimentin IFs. The deletion of PRD-B and PRD-C from the construct compromised its targeting to vimentin. Bioinformatic and structural modelling approaches provided multiple mechanisms by which the disease-causing mutations could potentially destabilise PRD structure and compromise cytoskeletal linkages. Overall, our data highlight potential molecular mechanisms underlying pathogenic missense mutations and could pave the way for informing novel curative interventions targeting cardiomyopathies and skin blistering disorders.
Assuntos
Desmoplaquinas/química , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Alelos , Substituição de Aminoácidos , Imunofluorescência , Estudos de Associação Genética , Predisposição Genética para Doença , Células HeLa , Humanos , Filamentos Intermediários/química , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Fenótipo , Proteínas Recombinantes , Solubilidade , Relação Estrutura-AtividadeRESUMO
Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.
Assuntos
Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/transmissão , COVID-19/virologia , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Fenômenos Biofísicos , COVID-19/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Simulação de Dinâmica Molecular , Mutação , Pandemias , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Homologia Estrutural de ProteínaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.
Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Antivirais/química , Evasão da Resposta Imune , Receptores de Coronavírus/química , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Deriva e Deslocamento Antigênicos , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de Coronavírus/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.
Assuntos
Mutação de Sentido Incorreto , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Modelos Moleculares , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.
Assuntos
Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Opsonização/imunologia , Engenharia de Proteínas , Sequência de Aminoácidos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ativação do Complemento , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Humanos , Fagocitose/imunologia , Ligação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Fc/genética , Proteína Estafilocócica A/imunologia , Staphylococcus aureus/imunologiaRESUMO
Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Células Cultivadas , Feminino , Células HEK293 , Humanos , Proteínas Relacionadas a Receptor de LDL/antagonistas & inibidores , Proteínas Relacionadas a Receptor de LDL/química , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/químicaRESUMO
Coronavirus Disease 2019 (COVID-19) is an ongoing global health emergency that has caused tremendous stress and loss of life worldwide. The viral spike glycoprotein is a critical molecule mediating transmission of SARS-CoV-2 by interacting with human ACE2. However, through the course of the pandemics, there has not been a thorough analysis of the spike protein mutations, and on how these mutants influence the transmission of SARS-CoV-2. Besides, cases of SARS-CoV-2 infection among pets and wild animals have been reported, so the susceptibility of these animals requires great attention to investigate, as they may also link to the renewed question of a possible intermediate host for SARS-CoV-2 before it was transmitted to humans. With over 226,000 SARS-CoV-2 sequences obtained, we found 1573 missense mutations in the spike gene, and 226 of them were within the receptor-binding domain (RBD) region that directly interacts with human ACE2. Modeling the interactions between SARS-CoV-2 spike mutants and ACE2 molecules showed that most of the 74 missense mutations in the RBD region of the interaction interface had little impact on spike binding to ACE2, whereas several within the spike RBD increased the binding affinity toward human ACE2 thus making the virus likely more contagious. On the other hand, modeling the interactions between animal ACE2 molecules and SARS-CoV-2 spike revealed that many pets and wild animals' ACE2 had a variable binding ability. Particularly, ACE2 of bamboo rat had stronger binding to SARS-CoV-2 spike protein, whereas that of mole, vole, Mus pahari, palm civet, and pangolin had a weaker binding compared to human ACE2. Our results provide structural insights into the impact on interactions of the SARS-CoV-2 spike mutants to human ACE2, and shed light on SARS-CoV-2 transmission in pets and wild animals, and possible clues to the intermediate host(s) for SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19/veterinária , COVID-19/virologia , Mutação de Sentido Incorreto , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/genética , Animais , Animais Selvagens/genética , Animais Selvagens/virologia , COVID-19/transmissão , Biologia Computacional , Interações entre Hospedeiro e Microrganismos/genética , Especificidade de Hospedeiro/genética , Humanos , Simulação de Dinâmica Molecular , Pandemias/veterinária , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Animais de Estimação/genética , Animais de Estimação/virologia , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de RiscoRESUMO
COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas contra COVID-19/imunologia , COVID-19/diagnóstico , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/genética , COVID-19/imunologia , Vacinas contra COVID-19/genética , Ensaios Clínicos como Assunto , Feminino , Engenharia Genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios e Motivos de Interação entre Proteínas/genética , Desenvolvimento de Vacinas , Vacinas de Subunidades Antigênicas/genéticaRESUMO
There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.
Assuntos
COVID-19/virologia , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , COVID-19/imunologia , COVID-19/transmissão , Evolução Molecular , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Moleculares , Pandemias , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.
Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/economia , Teste Sorológico para COVID-19/economia , Custos e Análise de Custo , Ensaio de Imunoadsorção Enzimática , Engenharia Genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
As protein-protein interactions (PPIs) are involved in many cellular events, development of mammalian cytosolic PPI detection systems is important for drug discovery as well as understanding biological phenomena. We have previously reported a c-kit-based PPI screening (KIPPIS) system, in which proteins of interest were fused with a receptor tyrosine kinase c-kit, leading to intracellular PPI-dependent cell growth. However, it has not been investigated whether PPI can be detected using other receptors. In this study, we employed a thrombopoietin receptor, which belongs to the Type I cytokine receptor family, to develop a thrombopoietin receptor-based PPI screening (THROPPIS) system. To improve the sensitivity of THROPPIS, we examined two strategies of (i) localization of the chimeric receptors on the cell membrane, and (ii) addition of a helper module to the chimeric receptors. Intriguingly, the nonlocalized chimeric receptor showed the best performance of THROPPIS. Furthermore, the addition of the helper module dramatically improved the detection sensitivity. In total, 5 peptide-domain interactions were detected successfully, demonstrating the versatility of THROPPIS. In addition, a peptide-domain interaction was detected even when insulin receptor or epidermal growth factor receptor was used as a signaling domain, demonstrating that this PPI detection system can be extended to other receptors.
Assuntos
Domínios e Motivos de Interação entre Proteínas/genética , Mapeamento de Interação de Proteínas/métodos , Receptores de Trombopoetina , Proteínas Recombinantes de Fusão , Animais , Linhagem Celular , Proliferação de Células/genética , Camundongos , Receptores de Trombopoetina/química , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genéticaRESUMO
Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.
Assuntos
Neoplasias/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Processamento U2AF/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Programas de Rastreamento , Simulação de Dinâmica Molecular , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Splicing de RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Spliceossomos/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
NF-Y is a pioneer transcription factor-TF-formed by the Histone-like NF-YB/NF-YC subunits and the regulatory NF-YA. It binds to the CCAAT box, an element enriched in promoters of genes overexpressed in many types of cancer. NF-YA is present in two major isoforms-NF-YAs and NF-YAl-due to alternative splicing, overexpressed in epithelial tumors. Here we analyzed NF-Y expression in stomach adenocarcinomas (STAD). We completed the partitioning of all TCGA tumor samples (450) according to molecular subtypes proposed by TCGA and ACRG, using the deep learning tool DeepCC. We analyzed differentially expressed genes-DEG-for enriched pathways and TFs binding sites in promoters. CCAAT is the predominant element only in the core group of genes upregulated in all subtypes, with cell-cycle gene signatures. NF-Y subunits are overexpressed, particularly NF-YA. NF-YAs is predominant in CIN, MSI and EBV TCGA subtypes, NF-YAl is higher in GS and in the ACRG EMT subtypes. Moreover, NF-YAlhigh tumors correlate with a discrete Claudinlow cohort. Elevated NF-YB levels are protective in MSS;TP53+ patients, whereas high NF-YAl/NF-YAs ratios correlate with worse prognosis. We conclude that NF-Y isoforms are associated to clinically relevant features of gastric cancer.
Assuntos
Fator de Ligação a CCAAT/genética , Regulação Neoplásica da Expressão Gênica , Subunidades Proteicas/genética , Neoplasias Gástricas/genética , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , TranscriptomaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.
Assuntos
SARS-CoV-2/genética , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/virologia , Genótipo , Humanos , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/classificação , Análise de Sequência de DNA/métodosRESUMO
Some recent SARS-CoV-2 variants appear to have increased transmissibility compared to the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on the target cells and infect them. In this study, we provide atomic-level insights into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities for the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than for the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , TermodinâmicaRESUMO
During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.