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CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis , Escherichia coli , Transferência Genética Horizontal , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may pre-order the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA binding specificity is robust to changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.
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CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d reveals that it does not require a protospacer flanking sequence but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is penalized by mismatches in the distal crRNA-target RNA region, while alterations in the proximal region inhibit nuclease activity. A biophysical model built from these data reveals that target recognition initiates in the distal end of the target RNA. Using this model, we design crRNAs that can differentiate between SARS-CoV-2 variants by modulating nuclease activation. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA , RNA/genética , RNA/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Endonucleases/metabolismoRESUMO
Type VI CRISPR enzymes cleave target RNAs and are widely used for gene regulation, RNA tracking, and diagnostics. However, a systematic understanding of their RNA binding specificity and cleavage activation is lacking. Here, we describe RNA chip-hybridized association-mapping platform (RNA-CHAMP), a massively parallel platform that repurposes next-generation DNA sequencing chips to measure the binding affinity for over 10,000 RNA targets containing structural perturbations, mismatches, insertions, and deletions relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d, a compact and widely used RNA nuclease, reveals that it does not require a protospacer flanking sequence (PFS) but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is strongly penalized by mismatches, insertions, and deletions in the distal crRNA-target RNA regions, while alterations in the proximal region inhibit nuclease activity without affecting binding. A biophysical model built from these data reveals that target recognition begins at the distal end of unstructured target RNAs and proceeds to the proximal end. Using this model, we designed a series of partially mismatched guide RNAs that modulate nuclease activity to detect single nucleotide polymorphisms (SNPs) in circulating SARS-CoV-2 variants. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme to improve CRISPR diagnostics and in vivo RNA editing. More broadly, RNA-CHAMP provides a quantitative platform for systematically measuring protein-RNA interactions.
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The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. For the Omicron variant, sub-lineages BA.1 and BA.2, respectively, contain 33 and 29 nonsynonymous and indel spike protein mutations. These amino acid substitutions and indels are implicated in increased transmissibility and enhanced immune evasion. By reverting individual spike mutations of BA.1 or BA.2, we characterize the molecular effects of the Omicron spike mutations on expression, ACE2 receptor affinity, and neutralizing antibody recognition. We identified key mutations enabling escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein can compensate for destabilizing mutations in the receptor binding domain, enabling the record number of mutations in Omicron. Our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illustrate previously uncharacterized mechanisms of host evasion.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais , Epitopos , Humanos , Glicoproteínas de Membrana , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope ViralRESUMO
The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions. Through combinatorial addition of disulfide bonds, cavity-filling substitutions, and improved electrostatic interactions, we describe a prefusion-stabilized F protein (DS-CavEs2) that expresses at 15 mg/L and has a melting temperature of 71.9 °C. Crystal structures of two prefusion-stabilized hMPV F variants reveal that antigenic surfaces are largely unperturbed. Importantly, immunization of mice with DS-CavEs2 elicits significantly higher neutralizing antibody titers against hMPV A1 and B1 viruses than postfusion F. The improved properties of DS-CavEs2 will advance the development of hMPV vaccines and the isolation of therapeutic antibodies.
Assuntos
Metapneumovirus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Imunização , Camundongos , Proteínas Virais de FusãoRESUMO
The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed â¼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.
Assuntos
Mamíferos/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Humanos , Mamíferos/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive ß-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.
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Anticorpos Antivirais/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Linhagem Celular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Reações Cruzadas , Desenho de Fármacos , Mapeamento de Epitopos , Feminino , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Vacinas Virais/imunologiaRESUMO
The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Afinidade de Anticorpos , COVID-19/prevenção & controle , Epitopos/imunologia , Humanos , Evasão da Resposta Imune , Imunoglobulina G/sangue , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Domínios Proteicos , Proteômica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.
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Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Anticorpos Neutralizantes/imunologia , Sequência de Bases , Betacoronavirus/imunologia , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , RNA-Polimerase RNA-Dependente de Coronavírus , Genoma Viral , Genótipo , Humanos , Aprendizado de Máquina , Modelos Moleculares , Técnicas de Diagnóstico Molecular , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Análise de Sequência de Proteína , Glicoproteína da Espícula de Coronavírus/imunologia , Texas/epidemiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.
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The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Assuntos
Substituição de Aminoácidos , Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Vacinas contra COVID-19 , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Humanos , Prolina/química , Domínios Proteicos , Estabilidade Proteica , SARS-CoV-2 , Vacinas Virais/químicaRESUMO
The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.
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The [10]phenacene and [11]phenacene molecules have been synthesized using a simple repetition of Wittig reactions followed by photocyclization. Sufficient amounts of [10]phenacene and [11]phenacene were obtained, and thin-film FETs using these molecules have been fabricated with SiO2 and ionic liquid gate dielectrics. These FETs operated in p-channel. The averaged measurements of field-effect mobility, <µ>, were 3.1(7) × 10-2 and 1.11(4) × 10-1 cm2 V-1 s-1, respectively, for [10]phenacene and [11]phenacene thin-film FETs with SiO2 gate dielectrics. Furthermore, [10]phenacene and [11]phenacene thin-film electric-double-layer (EDL) FETs with ionic liquid showed low-voltage p-channel FET properties, with <µ> values of 3(1) and 1(1) cm2 V-1 s-1, respectively. This study also discusses the future utility of the extremely extended π-network molecules [10]phenacene and [11]phenacene as the active layer of FET devices, based on the experimental results obtained.
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Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , RNA Interferente Pequeno , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A 4-Mbps 400-MHz On-Off Keying (OOK) receiver implemented in 0.18-um CMOS technology for implantable epilepsy sense-and-stimulation devices is presented. The proposed receiver is composed of a new current-mode full-wave envelope detector and differential cascaded gain amplifiers which is operated at MedRadio band. The fabricated receiver has power consumption of 0.27 mW and energy consumption of 0.07 nJ per bit at 4-Mbps. The sensitivity of receiver is -45.67 dBm.
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Amplificadores Eletrônicos , Processamento de Sinais Assistido por Computador/instrumentação , Telemetria/instrumentação , Algoritmos , Animais , Simulação por Computador , Fontes de Energia Elétrica , Epilepsia/terapia , Desenho de Equipamento , Humanos , Próteses e Implantes , Reprodutibilidade dos Testes , Semicondutores , Telemetria/métodos , Tecnologia sem FioRESUMO
Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase which promotes cell proliferation and survival, is abnormally overexpressed in numerous tumors of epithelial origin, including colorectal cancer (CRC). EGFR monoclonal antibodies have been shown to increase the median survival and are approved for the treatment of colorectal cancer. Histone deacetylases (HDACs), frequently overexpressed in colorectal cancer and several malignancies, are another attractive targets for cancer therapy. Several inhibitors of HDACs (HDACi) are developed and exhibit powerful antitumor abilities. In this study, human colorectal cancer cells treated with HDACi exhibited reduced EGFR expression, thereby disturbed EGF-induced ERK and Akt phosphorylation. HDACi also decreased the expression of SGLT1, an active glucose transporter found to be stabilized by EGFR, and suppressed the glucose uptake of cancer cells. HDACi suppressed the transcription of EGFR and class I HDACs were proved to be involved in this event. Chromatin immunoprecipitation analysis showed that HDACi caused the dissociation of SP1, HDAC3 and CBP from EGFR promoter. Our data suggested that HDACi could serve as a single agent to block both EGFR and HDAC, and may bring more benefits to the development of CRC therapy.
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Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Inativação Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/metabolismo , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Elevated levels of NF-kappaB are frequently detected in many inflammatory diseases and cancers. Blocking the IKK-NF-kappaB pathway has been seen as a promising approach for new therapies. By employing the dominant-negative mutant of IKKbeta, our data revealed that loss of IKKbeta activity reduces not only the proliferation and invasion of lung adenocarcinoma A549 cells in vitro but also the tumour formation, metastasis and angiogenesis in mouse xenograft model. Treatment of IKKbeta inhibitors (CYL-19s and CYL-26z) leads to the arrest of cell cycle progression at G1 and G2/M, followed by apoptosis. IKKbeta inhibitors can increase the protein stability, nuclear accumulation and promoter-binding activity of p53, leading to the p21 gene transcription. Furthermore, knockdown of IKKbeta by siRNA increased the stability and expression of p53 and p21 promoter activity. In addition, IKKbeta inhibitor-induced p53 and p21 expressions were augmented in the presence of IKKbeta siRNA. Correlation between p53 acetylation and its protein stabilization was also seen after treatment with IKKbeta inhibitors. These results suggest that loss of IKKbeta activation is important for the enhancement of p53 stability, leading to p21 expression and cell cycle arrest and apoptosis of tumour cells.
Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinase I-kappa B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Furanos/farmacologia , Células HCT116 , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Proteína Supressora de Tumor p53/genéticaRESUMO
TGF-beta regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-beta has been reported to mediate stimulatory action of TGF-beta-induced ECM. Although TNF-alpha was reported to suppress theTGF-beta-induced CTGF gene expression, the molecular mechanism is not well clarified. In this study, we found the inhibitory effect of TNF-alpha on TGF-beta-induced CTGF expression in WT but not p65-/-MEF cells. TNF-alpha neither induced Smad7 expression nor affected TGF-beta-induced Smad2 phosphorylation and nuclear translocation. We demonstrated that p300 physically associated with p65 rather than Smad4 in the presence of both TNF-alpha and TGF-beta. Moreover, the TGF-beta-induced binding of p300 and acetylated H4, but not Smad4 to the CTGF promoter was disturbed by TNF-alpha treatment. Overall, our data showed that suppression of TNF-alpha on TGF-beta-induced CTGF expression is due to the competition of p300 by p65 and Smad4.