RESUMEN
Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a â¼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.
Asunto(s)
Neoplasias Hematológicas , Fosfoproteínas , Elongación de la Transcripción Genética , Factores de Transcripción , Humanos , Neoplasias Hematológicas/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Fosfoproteínas/genéticaRESUMEN
Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.
Asunto(s)
Proteínas Fúngicas , Oryza , Enfermedades de las Plantas , Fosforilación , Oryza/microbiología , Oryza/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Ascomicetos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteómica , Transducción de SeñalRESUMEN
Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.
Asunto(s)
Linfocitos T CD8-positivos , Proteínas de Unión al ADN , Interferón Tipo I , Proteínas de la Membrana , Neoplasias , Transducción de Señal , Factores de Transcripción , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Mutación , Neoplasias/inmunología , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Masculino , Quimiocinas/genética , Quimiocinas/metabolismoRESUMEN
Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.
Asunto(s)
Virus de la Parotiditis , Infección Persistente , Humanos , Virus de la Parotiditis/fisiología , Nucleocápside , Fosfoproteínas/metabolismo , Replicación ViralRESUMEN
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Daño del ADN , Exodesoxirribonucleasas/metabolismo , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , Animales , Línea Celular , Senescencia Celular , Colágeno/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Invasividad Neoplásica , Membrana Nuclear/ultraestructura , Proteolisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteogenómica , Adenocarcinoma/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Carcinoma Ductal Pancreático/diagnóstico , Estudios de Cohortes , Células Endoteliales/metabolismo , Epigénesis Genética , Femenino , Dosificación de Gen , Genoma Humano , Glucólisis , Glicoproteínas/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Neoplasias Pancreáticas/diagnóstico , Fenotipo , Fosfoproteínas/metabolismo , Fosforilación , Pronóstico , Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Especificidad por Sustrato , Transcriptoma/genéticaRESUMEN
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Teléfono Celular/instrumentación , Imagen Óptica/métodos , ARN Viral/análisis , Carga Viral/métodos , Animales , Prueba de Ácido Nucleico para COVID-19/economía , Prueba de Ácido Nucleico para COVID-19/instrumentación , Sistemas CRISPR-Cas , Línea Celular , Proteínas de la Nucleocápside de Coronavirus/genética , Humanos , Nasofaringe/virología , Imagen Óptica/instrumentación , Fosfoproteínas/genética , Pruebas en el Punto de Atención , Interferencia de ARN , ARN Viral/genética , Sensibilidad y Especificidad , Carga Viral/economía , Carga Viral/instrumentaciónRESUMEN
Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos T , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Receptores de Antígenos de Linfocitos T/metabolismo , Activación de Linfocitos , Fosforilación , Fosfoproteínas/genéticaRESUMEN
We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteogenómica , Neoplasias Encefálicas/inmunología , Niño , Variaciones en el Número de Copia de ADN/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genoma Humano , Glioma/genética , Glioma/patología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Mutación/genética , Clasificación del Tumor , Recurrencia Local de Neoplasia/patología , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma/genéticaRESUMEN
Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90ß as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Proteómica , Adenocarcinoma del Pulmón/genética , Pueblo Asiatico/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Sistemas de Liberación de Medicamentos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Estadificación de Neoplasias , Fosfoproteínas/metabolismo , Análisis de Componente Principal , Pronóstico , Proteoma/metabolismo , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genéticaRESUMEN
To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.
Asunto(s)
Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteogenómica , Adenocarcinoma del Pulmón/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Masculino , Persona de Mediana Edad , Mutación/genética , Proteínas de Fusión Oncogénica , Fenotipo , Fosfoproteínas/metabolismo , Proteoma/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Sistemas CRISPR-Cas , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Células A549 , Profilaxis Antibiótica/métodos , Secuencia de Bases , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Simulación por Computador , Secuencia Conservada , Coronavirus/efectos de los fármacos , Coronavirus/genética , Coronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/tratamiento farmacológico , Proteínas de la Nucleocápside de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , Células Epiteliales/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Pulmón/patología , Pulmón/virología , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , Filogenia , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2 , Proteínas no Estructurales Virales/genéticaRESUMEN
The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.
Asunto(s)
COVID-19/inmunología , Inmunidad Humoral , Receptores de Reconocimiento de Patrones/inmunología , SARS-CoV-2/inmunología , Animales , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , COVID-19/metabolismo , COVID-19/virología , Estudios de Casos y Controles , Chlorocebus aethiops , Activación de Complemento , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Femenino , Glicosilación , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Masculino , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Lectina de Unión a Manosa/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Polimorfismo Genético , Unión Proteica , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Componente Amiloide P Sérico/inmunología , Componente Amiloide P Sérico/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.
Asunto(s)
Fosfoproteínas/ultraestructura , ARN Polimerasa Dependiente del ARN/ultraestructura , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/enzimología , Proteínas Virales/ultraestructura , Acetatos/química , Animales , Antivirales/química , Antivirales/uso terapéutico , Dominio Catalítico , Microscopía por Crioelectrón , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Quinolinas/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Vacunas contra Virus Sincitial Respiratorio/química , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
Asunto(s)
Carcinogénesis/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animales , Humanos , Espectrometría de Masas/métodos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteómica , Ratas , Ratas Sprague-Dawley , Pez CebraRESUMEN
Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.
Asunto(s)
Uniones Estrechas/genética , Proteínas de la Zonula Occludens/genética , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-2/genética , Animales , Sitios de Unión/genética , Adhesión Celular/genética , Polaridad Celular/genética , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Dominios PDZ/genética , Fosfoproteínas/genética , Fosforilación/genética , Unión Proteica/genética , Transducción de Señal/genética , Uniones Estrechas/metabolismo , Proteínas de la Zonula Occludens/química , Proteínas de la Zonula Occludens/ultraestructura , Proteína de la Zonula Occludens-1/química , Proteína de la Zonula Occludens-1/ultraestructura , Proteína de la Zonula Occludens-2/química , Proteína de la Zonula Occludens-2/ultraestructura , Dominios Homologos src/genéticaRESUMEN
Cell-cell junctions respond to mechanical forces by changing their organization and function. To gain insight into the mechanochemical basis underlying junction mechanosensitivity, we analyzed tight junction (TJ) formation between the enveloping cell layer (EVL) and the yolk syncytial layer (YSL) in the gastrulating zebrafish embryo. We found that the accumulation of Zonula Occludens-1 (ZO-1) at TJs closely scales with tension of the adjacent actomyosin network, revealing that these junctions are mechanosensitive. Actomyosin tension triggers ZO-1 junctional accumulation by driving retrograde actomyosin flow within the YSL, which transports non-junctional ZO-1 clusters toward the TJ. Non-junctional ZO-1 clusters form by phase separation, and direct actin binding of ZO-1 is required for stable incorporation of retrogradely flowing ZO-1 clusters into TJs. If the formation and/or junctional incorporation of ZO-1 clusters is impaired, then TJs lose their mechanosensitivity, and consequently, EVL-YSL movement is delayed. Thus, phase separation and flow of non-junctional ZO-1 confer mechanosensitivity to TJs.
Asunto(s)
Desarrollo Embrionario/genética , Mecanotransducción Celular/genética , Uniones Estrechas/genética , Proteína de la Zonula Occludens-1/genética , Citoesqueleto de Actina/genética , Actomiosina/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Fosfoproteínas/genética , Unión Proteica , Uniones Estrechas/fisiología , Saco Vitelino/crecimiento & desarrollo , Saco Vitelino/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Línea Celular , Autorrenovación de las Células , Inmunoprecipitación de Cromatina , Retrovirus Endógenos/genética , Femenino , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Oligorribonucleótidos Antisentido/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Interferencia de ARN , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Regulación hacia Arriba , NucleolinaRESUMEN
Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.
Asunto(s)
Hipoxia de la Célula , Relojes Circadianos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Aminoácidos Dicarboxílicos/farmacología , Animales , Proteínas CLOCK/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Relojes Circadianos/efectos de los fármacos , Medios de Cultivo/química , Factores Eucarióticos de Iniciación , Concentración de Iones de Hidrógeno , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/metabolismo , Transcriptoma/efectos de los fármacos , Proteína 2 del Complejo de la Esclerosis Tuberosa/deficiencia , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.