RESUMEN
A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.
Asunto(s)
Complejo CD3/metabolismo , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Complejo CD3/química , Proteína Tirosina Quinasa CSK/metabolismo , Línea Celular , Citocinas/metabolismo , Humanos , Activación de Linfocitos/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Ratones Endogámicos NOD , Neoplasias/mortalidad , Neoplasias/patología , Neoplasias/terapia , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Supervivencia , Vanadatos/farmacologíaRESUMEN
Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.
Asunto(s)
Antibacterianos/farmacología , Anticuerpos Antivirales/inmunología , Microbioma Gastrointestinal/fisiología , Inmunidad/efectos de los fármacos , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Adolescente , Adulto , Formación de Anticuerpos , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Voluntarios Sanos , Humanos , Inmunogenicidad Vacunal/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Masculino , Adulto JovenRESUMEN
Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Código de Histonas/efectos de los fármacos , Código de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Activación Transcripcional , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Factores de Transcripción p300-CBP/fisiologíaRESUMEN
Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Gripe Humana/patología , Neuraminidasa/inmunología , Proteínas Virales/inmunología , Animales , Aves , Reacciones Cruzadas , Epítopos/inmunología , Femenino , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
SARS-CoV-2 Omicron variant has presented significant challenges to current antibodies and vaccines. Herein, we systematically compared the efficacy of 50 human monoclonal antibodies (mAbs), covering the seven identified epitope classes of the SARS-CoV-2 RBD, against Omicron sub-variants BA.1, BA.1.1, BA.2, and BA.3. Binding and pseudovirus-based neutralizing assays revealed that 37 of the 50 mAbs lost neutralizing activities, whereas the others displayed variably decreased activities against the four Omicron sub-variants. BA.2 was found to be more sensitive to RBD-5 antibodies than the other sub-variants. Furthermore, a quaternary complex structure of BA.1 RBD with three mAbs showing different neutralizing potencies against Omicron provided a basis for understanding the immune evasion of Omicron sub-variants and revealed the lack of G446S mutation accounting for the sensitivity of BA.2 to RBD-5 mAbs. Our results may guide the application of the available mAbs and facilitate the development of universal therapeutic antibodies and vaccines against COVID-19.
Asunto(s)
Anticuerpos Neutralizantes , COVID-19 , Anticuerpos Monoclonales , Anticuerpos Antivirales , Vacunas contra la COVID-19 , Humanos , Glicoproteínas de Membrana , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio ViralRESUMEN
DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Daño del ADN , Humanos , Citometría de Flujo , Transducción de Señal , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Genoma , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genéticaRESUMEN
Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.
Asunto(s)
Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , COVID-19/inmunología , Interacciones Huésped-Patógeno/inmunología , Epítopos Inmunodominantes/inmunología , SARS-CoV-2/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/genética , Linfocitos B/metabolismo , Biología Computacional/métodos , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Humanos , Epítopos Inmunodominantes/genética , Memoria Inmunológica , Masculino , Pruebas de Neutralización , Análisis de la Célula Individual/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , TranscriptomaRESUMEN
Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding RNAs (lncRNAs) have been implicated in tumor drug resistance; however, their potential significance in DDR is still largely unknown. Here, we report that a human lncRNA, CTD-2256P15.2, encodes a micropeptide, named PAR-amplifying and CtIP-maintaining micropeptide (PACMP), with a dual function to maintain CtIP abundance and promote poly(ADP-ribosyl)ation. PACMP not only prevents CtIP from ubiquitination through inhibiting the CtIP-KLHL15 association but also directly binds DNA damage-induced poly(ADP-ribose) chains to enhance PARP1-dependent poly(ADP-ribosyl)ation. Targeting PACMP alone inhibits tumor growth by causing a synthetic lethal interaction between CtIP and PARP inhibitions and confers sensitivity to PARP/ATR/CDK4/6 inhibitors, ionizing radiation, epirubicin, and camptothecin. Our findings reveal that a lncRNA-derived micropeptide regulates cancer progression and drug resistance by modulating DDR, whose inhibition could be employed to augment the existing anticancer therapeutic strategies.
Asunto(s)
Endodesoxirribonucleasas , Neoplasias , Péptidos , Poli ADP Ribosilación , ARN Largo no Codificante , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Péptidos/farmacología , Poli Adenosina Difosfato Ribosa/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.
Asunto(s)
Proteómica , Ubiquitina Tiolesterasa , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteoma , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.
Asunto(s)
Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Orthomyxoviridae/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Anticuerpos ampliamente neutralizantes/genética , Reacciones Cruzadas , Epítopos de Linfocito B/inmunología , Genes de Inmunoglobulinas , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Orthomyxoviridae/clasificación , Dominios Proteicos , Hipermutación Somática de InmunoglobulinaRESUMEN
KRAS mutant cancer, characterized by the activation of a plethora of phosphorylation signaling pathways, remains a major challenge for cancer therapy. Despite recent advancements, a comprehensive profile of the proteome and phosphoproteome is lacking. This study provides a proteomic and phosphoproteomic landscape of 43 KRAS mutant cancer cell lines across different tissue origins. By integrating transcriptomics, proteomics, and phosphoproteomics, we identify three subsets with distinct biological, clinical, and therapeutic characteristics. The integrative analysis of phosphoproteome and drug sensitivity information facilitates the identification of a set of drug combinations with therapeutic potentials. Among them, we demonstrate that the combination of DOT1L and SHP2 inhibitors is an effective treatment specific for subset 2 of KRAS mutant cancers, corresponding to a set of TCGA clinical tumors with the poorest prognosis. Together, this study provides a resource to better understand KRAS mutant cancer heterogeneity and identify new therapeutic possibilities.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores Enzimáticos/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Fosfoproteínas/metabolismo , Proteoma , Proteómica , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Línea Celular Tumoral , Bases de Datos Genéticas , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Espectrometría de Masas , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosfoproteínas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Anticuerpos ampliamente neutralizantes , Epítopos , Glicoproteínas Hemaglutininas del Virus de la Influenza , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Epítopos/química , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Células B de Memoria/inmunologíaRESUMEN
Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6(-/-) embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6(+/-)) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.
Asunto(s)
Inflamación/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Masculino , Ratones , Persona de Mediana Edad , Sepsis/sangre , Choque Séptico/inducido químicamente , Choque Séptico/metabolismoRESUMEN
The 26S proteasome is the ATP-dependent protease responsible for regulating the proteome of eukaryotic cells through degradation of mainly ubiquitin-tagged substrates. In order to understand how proteasome responds to ubiquitin signal, we resolved an ensemble of cryo-EM structures of proteasome in the presence of K48-Ub4, with three of them resolved at near-atomic resolution. We identified a conformation with stabilized ubiquitin receptors and a previously unreported orientation of the lid, assigned as a Ub-accepted state C1-b. We determined another structure C3-b with localized K48-Ub4 to the toroid region of Rpn1, assigned as a substrate-processing state. Our structures indicate that tetraUb induced conformational changes in proteasome could initiate substrate degradation. We also propose a CP gate-opening mechanism involving the propagation of the motion of the lid to the gate through the Rpn6-α2 interaction. Our results enabled us to put forward a model of a functional cycle for proteasomes induced by tetraUb and nucleotide.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Ubiquitina/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/ultraestructura , Unión Proteica , Conformación Proteica , Proteolisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Relación Estructura-Actividad , Ubiquitina/ultraestructura , UbiquitinaciónRESUMEN
Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.
Asunto(s)
Comunicación Celular , Pollos , Infecciones por Coronavirus , Redes Reguladoras de Genes , Virus de la Bronquitis Infecciosa , Análisis de la Célula Individual , Animales , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/fisiología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/genética , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/inmunología , Análisis de Secuencia de ARN , Células Epiteliales/virología , Células Epiteliales/metabolismoRESUMEN
Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines.
Asunto(s)
Pollos , Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Glicoproteína de la Espiga del Coronavirus , Vacunas Atenuadas , Animales , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/patogenicidad , Embrión de Pollo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Vacunas Atenuadas/genética , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/genética , Virulencia , Vacunas Virales/genética , Vacunas Virales/inmunología , MutaciónRESUMEN
Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.
Asunto(s)
Hidrocefalia , Escoliosis , Urotensinas , Animales , Cilios/metabolismo , Epéndimo/metabolismo , Epéndimo/patología , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hidrocefalia/patología , Escoliosis/genética , Escoliosis/metabolismo , Escoliosis/patología , Urotensinas/metabolismo , Pez CebraRESUMEN
Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule. It is also a critical player in the regulation of cell size and cell behaviors such as cell aggregation and phototaxis in cyanobacteria, which constitute an important group of prokaryotes for their roles in the ecology and evolution of the Earth. However, c-di-GMP receptors have never been revealed in cyanobacteria. Here, we report the identification of a c-di-GMP receptor, CdgR, from the filamentous cyanobacterium Anabaena PCC 7120. Crystal structural analysis and genetic studies demonstrate that CdgR binds c-di-GMP at the dimer interface and this binding is required for the control of cell size in a c-di-GMP-dependent manner. Different functions of CdgR, in ligand binding and signal transmission, could be separated genetically, allowing us to dissect its molecular signaling functions. The presence of the apo-form of CdgR triggers cell size reduction, consistent with the similar effects observed with a decrease of c-di-GMP levels in cells. Furthermore, we found that CdgR exerts its function by interacting with a global transcription factor DevH, and this interaction was inhibited by c-di-GMP. The lethal effect triggered by conditional depletion of DevH or by the production of several point-mutant proteins of CdgR in cells indicates that this signaling pathway plays critical functions in Anabaena. Our studies revealed a mechanism of c-di-GMP signaling in the control of cell size, an important and complex trait for bacteria. CdgR is highly conserved in cyanobacteria, which will greatly expand our understanding of the roles of c-di-GMP signaling in these organisms.
Asunto(s)
Cianobacterias , Transducción de Señal , Cianobacterias/metabolismo , GMP Cíclico/metabolismo , Regulación de la Expresión Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
The recent development of solvent- and polymer-based brain-clearing techniques has advanced our ability to visualize the mammalian nervous system in three dimensions. However, it remains challenging to image the mammalian body en bloc. Here we developed HYBRiD (hydrogel-based reinforcement of three-dimensional imaging solvent-cleared organs (DISCO)), by recombining components of organic- and polymer-based clearing pipelines. We achieved high transparency and protein retention, as well as compatibility with direct fluorescent imaging and immunostaining in cleared mammalian bodies. Using parvalbumin- and somatostatin-Cre models, we demonstrated the utility of HYBRiD for whole-body imaging of genetically encoded fluorescent reporters without antibody enhancement of signals in newborn and juvenile mice. Using K18-hACE2 transgenic mice, HYBRiD enabled perfusion-free clearing and visualization of SARS-CoV-2 infection in a whole mouse chest, revealing macroscopic and microscopic features of viral pathology in the same sample. HYBRiD offers a simple and universal solution to visualize large heterogeneous body parts or entire animals for basic and translational research.
Asunto(s)
COVID-19 , Hidrogeles , Animales , Imagenología Tridimensional/métodos , Mamíferos , Ratones , Polímeros , SARS-CoV-2 , SolventesRESUMEN
Kinesin motors play a fundamental role in development by controlling intracellular transport, spindle assembly, and microtubule organization. In humans, patients carrying mutations in KIF11 suffer from an autosomal dominant inheritable disease called microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR). While mitotic functions of KIF11 proteins have been well documented in centrosome separation and spindle assembly, cellular mechanisms underlying KIF11 dysfunction and MCLMR remain unclear. In this study, we generate KIF11-inhibition chick and zebrafish models and find that KIF11 inhibition results in microcephaly, chorioretinopathy, and severe developmental defects in vivo. Notably, loss-of-function of KIF11 causes the formation of monopolar spindle and chromosome misalignment, which finally contribute to cell cycle arrest, chromosome instability, and cell death. Our results demonstrate that KIF11 is crucial for spindle assembly, chromosome alignment, and cell cycle progression of progenitor stem cells, indicating a potential link between polyploidy and MCLMR. Our data have revealed that KIF11 inhibition cause microcephaly, chorioretinopathy, and development disorders through the formation of monopolar spindle, polyploid, and cell cycle arrest.