RESUMEN
SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.
Asunto(s)
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , ARN Viral/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Interferones/genética , CitocinasRESUMEN
RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.
Asunto(s)
Proteína de Unión al Tracto de Polipirimidina , ARN , Humanos , ARN/metabolismo , Células HeLa , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme Alternativo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , ARN/genética , Análisis de Secuencia de ARN/métodos , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Cromosomas Humanos/genética , Elementos de Facilitación Genéticos/genética , Genes myc/genética , Genes de ARNr/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
Sialic acid-binding Ig-like lectin-15 is an important immunosuppressive molecule considered to be a key target in next-generation tumor immunotherapy. In this study, we screened 22 high-affinity antibodies that specifically recognize human Siglec-15 by using a large human phage antibody library, and five representative sequences were selected for further study. The results showed the binding activity of five antibodies to Siglec-15 (EC50 ranged from 0.02368 µg/mL to 0.07949 µg/mL), and in two Siglec-15-overexpressed cell lines, three antibodies had the strongest binding activity, so the two clones were discarded for further study. Subsequently, the affinity of three antibodies were measured by bio-layer interferometry technology (5-9 × 10E-09M). As the reported ligands of Siglec-15, the binding activity of Siglec-15 and sialyl-Tn, cluster of differentiation 44, myelin-associated glycoprotein, and leucine-rich repeat-containing protein 4C can be blocked by three of the antibodies. Among these, 3F1 had a competitive advantage. Then, the antibody 3F1 showed an obvious antibody-dependent cell-mediated cytotoxicity effect (EC50 was 0.85 µg/mL). Further, antibody 3F1 can reverse the inhibitory effect of Siglec-15 on lymphocyte proliferation (especially CD4+T and CD8+T) and cytokine release Interferon-γ. Given the above results, 3F1 was selected as a candidate for the in vivo pharmacodynamics study. In the tumor model of Balb/c Nude mice, 3F1 (10 mg/kg) showed certain antitumor effects [tumor growth inhibition (TGI) was 31.5%], while the combination of 3F1 (5 mg/kg) and Erbitux (5 mg/kg) showed significant antitumor effects (TGI was 48.7%) compared with the PBS group. In conclusion, novel human antibody 3F1 has antitumor activity and is expected to be an innovative candidate drug targeting Siglec-15 for tumor immunotherapy. SIGNIFICANCE STATEMENT: Siglec-15 is considered as an important target in the next generation of tumor immunotherapy. 3F1 is expected to be the most promising potential candidate for targeting Siglec-15 for cancer treatment and could provide a reference for the development of antitumor drugs.
Asunto(s)
Antígenos CD , Neoplasias , Animales , Antígenos CD/metabolismo , Humanos , Inmunoglobulinas , Lectinas/química , Lectinas/metabolismo , Ligandos , Proteínas de la Membrana , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológicoRESUMEN
Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Diferenciación/efectos adversos , Antígenos de Diferenciación/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Antígeno CD47/química , Células CHO , Línea Celular , Cricetulus , Diseño de Fármacos , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Hemaglutinación/efectos de los fármacos , Inmunoterapia , Ratones SCID , Modelos Moleculares , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Rituximab/uso terapéutico , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Staphylococcal enterotoxin B (SEB), one of the exotoxins produced by Staphylococcus aureus, is the key toxin that causes poisoning reactions and toxic shock syndrome. In the current research work, a novel human antibody named LXY8 was screened from a human phage display antibody library, and LXY8 blocked the interaction between SEB and the T cell receptor (TCR). The binding activity between LXY8 and SEB was 0.525 nM. Furthermore, LXY8 could effectively inhibit the SEB-induced activation of peripheral blood mononuclear cells and release of cytokines. In the BALB/c mouse model, LXY8 effectively neutralized SEB toxicity in vivo. Finally, based on computer-guided molecular modeling, we designed a series of SEB mutation sites; these sites facilitated the determination of the key residues (i.e.176EFNN179) of SEB recognized by LXY8. The research revealed that the 176EFNN179 residues of SEB are important for specific antibody-antigen recognition. The results may be helpful for the development of antibody-based therapy for SEB-induced toxic shock syndrome.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Anticuerpos Monoclonales/análisis , Anticuerpos Neutralizantes/análisis , Enterotoxinas/inmunología , Epítopos/inmunología , Animales , Células CHO , Proliferación Celular , Técnicas de Visualización de Superficie Celular , Cricetulus , Citocinas/metabolismo , Enterotoxinas/antagonistas & inhibidores , Mapeo Epitopo , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones Endogámicos BALB C , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
RNA-binding proteins (RBPs) can bind and mediate RNA-RNA contacts. However, identifying specific RBP-organized RNA-RNA contacts remains challenging. Here, we present a capture RIC-seq (CRIC-seq) technique to map specific RBP-associated RNA-RNA contacts globally. We describe steps for formaldehyde cross-linking to fix RNA in situ conformation, pCp-biotin labeling to mark RNA juncture, and in situ proximity ligation to join proximal RNAs. We then detail immunoprecipitation to isolate specific RBP-associated RNA-RNA contacts, biotin-streptavidin selection to enrich chimeric RNAs, and library construction for paired-end sequencing. For complete information on the generation and use of this protocol, please refer to Ye et al.1.
Asunto(s)
Biotina , ARN , ARN/química , Biotina/metabolismo , Proteínas de Unión al ARN/metabolismo , Inmunoprecipitación , Estreptavidina/metabolismoRESUMEN
CD47, as an innate immune checkpoint molecule, is an important target of cancer immunotherapy. We previously reported that a high-affinity SIRPα variant FD164 fused with IgG1 subtype Fc showed a better antitumor effect than wild-type SIRPα in an immunodeficient tumor-bearing model. However, CD47 is widely expressed in blood cells, and the drugs targeting CD47 may cause potential hematological toxicity. Herein, we modified the FD164 molecule by Fc mutation (N297A) to inactivate the Fc-related effector function and named it nFD164. Moreover, we further studied the potential of nFD164 as a candidate drug targeting CD47, including the stability, in vitro activity, antitumor activity of single or combined drugs in vivo, and hematological toxicity in humanized CD47/SIRPα transgenic mouse model. The results show that nFD164 maintains strong binding activity to CD47 on tumor cells, but has weak binding activity with red blood cells or white blood cells, and nFD164 has good drug stability under accelerated conditions (high temperature, bright light and freeze-thaw cycles). More importantly, in the immunodeficient or humanized CD47/SIRPα transgenic mice bearing tumor model, the combination of nFD164 and anti-CD20 antibody or anti-mPD-1 antibody had a synergistic antitumor effect. Especially in transgenic mouse models, nFD164 combined with anti-mPD-1 significantly enhanced tumor suppressive activity compared with anti-mPD-1 (P < 0.01) or nFD164 (P < 0.01) as a single drug and had fewer hematology-related side effects than FD164 or Hu5F9-G4. When these factors are taken together, nFD164 is a promising high-affinity CD47-targeting drug candidate with better stability, potential antitumor activity, and improved safety profile.
Asunto(s)
Antígeno CD47 , Neoplasias , Ratones , Animales , Antígeno CD47/metabolismo , Inmunoterapia/métodos , Modelos Animales de Enfermedad , Ratones Transgénicos , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , FagocitosisRESUMEN
Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.
RESUMEN
The continuous mutation of SARS-CoV-2 has presented enormous challenges to global pandemic prevention and control. Recent studies have shown evidence that the genome sequence of SARS-CoV-2 nucleocapsid proteins is relatively conserved, and their biological functions are being confirmed. There is increasing evidence that the N protein will not only provide a specific diagnostic marker but also become an effective treatment target. In this study, 2G4, which specifically recognizes the N protein, was identified by screening a human phage display library. Based on the computer-guided homology modelling and molecular docking method used, the 3-D structures for the 2G4 scFv fragment (VH-linker-VL structure, with (G4S)3 as the linker peptide in the model), SARS-CoV-2 N protein and its complex were modelled and optimized with a suitable force field. The binding mode and key residues of the 2G4 and N protein interaction were predicted, and three mutant antibodies (named 2G4-M1, 2G4-M2 and 2G4-M3) with higher affinity were designed theoretically. Using directed point mutant technology, the three mutant antibodies were prepared, and their affinity was tested. Their affinity constants of approximately 0.19 nM (2G4-M1), 0.019 nM (2G4-M2) and 0.075 nM (2G4-M3) were at least one order of magnitude lower than that of the parent antibody (3 nM; 2G4, parent antibody), as determined using a biolayer interferometry (BLI) assay. It is expected that high-affinity candidates will be used for diagnosis and even as potential therapeutic drugs for the SARS-CoV-2 pandemic.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales , Afinidad de Anticuerpos , Técnicas de Visualización de Superficie Celular , Humanos , Simulación del Acoplamiento Molecular , SARS-CoV-2/genéticaRESUMEN
Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3' end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.
Asunto(s)
ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Animales , HumanosRESUMEN
SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill the knowledge gap and facilitate structure-based drug development, we develop a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstruct the tertiary structure of the SARS-CoV-2 genome and reveal a surprisingly "unentangled globule" conformation. We uncover many long-range duplexes and higher-order junctions, both of which are under purifying selections and contribute to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations may remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs can obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.
Asunto(s)
COVID-19/patología , ARN Viral/química , SARS-CoV-2/genética , Análisis de Secuencia de ARN/métodos , Virión/genética , Animales , COVID-19/genética , COVID-19/virología , Células Cultivadas , Chlorocebus aethiops , Genoma Viral , Humanos , Conformación de Ácido Nucleico , ARN Viral/genética , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidad , Virión/química , Virión/metabolismoRESUMEN
A series of novel linear aliphatic amine-linked triaryl derivatives as inhibitors of PD-1/PD-L1 were designed, synthesized, and evaluated in vitro and in vivo. In this chemical series, compound 58 showed the most potent inhibitory activity and binding affinity with hPD-L1, with an IC50 value of 12 nM and a KD value of 16.2 pM, showing a binding potency approximately 2000-fold that of hPD-1. Compound 58 could bind with hPD-L1 on the cellular surface and competitively block the interaction of hPD-1 with hPD-L1. In a T cell function assay, 58 restored the T cell function, leading to increased IFN-γ secretion. Moreover, in a humanized mouse model, compound 58 significantly inhibited tumor growth without obvious toxicity and showed moderate PK properties after intravenous injection. These results indicated that 58 is a promising lead for further development of small-molecule PD-1/PD-L1 inhibitors for cancer therapy.