RESUMEN
The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.
Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Neumonía Viral/metabolismo , Proteómica/métodos , Células A549 , Enzima Convertidora de Angiotensina 2 , Animales , Antivirales/farmacología , COVID-19 , Células CACO-2 , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Fosforilación , Neumonía Viral/virología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
Asunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/metabolismo , Reposicionamiento de Medicamentos , Terapia Molecular Dirigida , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/metabolismo , Mapas de Interacción de Proteínas , Proteínas Virales/metabolismo , Animales , Antivirales/clasificación , Antivirales/farmacología , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidad , COVID-19 , Chlorocebus aethiops , Clonación Molecular , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad Innata , Espectrometría de Masas , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Dominios Proteicos , Mapeo de Interacción de Proteínas , Receptores sigma/metabolismo , SARS-CoV-2 , Proteínas Ligasas SKP Cullina F-box/metabolismo , Células Vero , Proteínas Virales/genética , Tratamiento Farmacológico de COVID-19RESUMEN
Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
Asunto(s)
Microscopía por Crioelectrón , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Transporte Activo de Núcleo Celular , Secuencia de Bases , Dominio Catalítico , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citoplasma/metabolismo , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , ADN Espaciador Ribosómico/metabolismo , ADN Espaciador Ribosómico/ultraestructura , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/ultraestructura , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Unión Proteica , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Hongos/ultraestructura , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/aislamiento & purificación , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Rotación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large subunit rRNAs and a number of ancillary "glue" proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.
Asunto(s)
Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Ribosomas/ultraestructura , Trypanosoma cruzi/química , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , ARN Ribosómico/genética , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/genética , Ribosomas/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestructuraRESUMEN
Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo-that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling.
Asunto(s)
Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purificationmass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Espectrometría de Masas , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Purificación por Afinidad en TándemRESUMEN
A major goal of cancer research is to understand how mutations distributed across diverse genes affect common cellular systems, including multiprotein complexes and assemblies. Two challengeshow to comprehensively map such systems and how to identify which are under mutational selectionhave hindered this understanding. Accordingly, we created a comprehensive map of cancer protein systems integrating both new and published multi-omic interaction data at multiple scales of analysis. We then developed a unified statistical model that pinpoints 395 specific systems under mutational selection across 13 cancer types. This map, called NeST (Nested Systems in Tumors), incorporates canonical processes and notable discoveries, including a PIK3CA-actomyosin complex that inhibits phosphatidylinositol 3-kinase signaling and recurrent mutations in collagen complexes that promote tumor proliferation. These systems can be used as clinical biomarkers and implicate a total of 548 genes in cancer evolution and progression. This work shows how disparate tumor mutations converge on protein assemblies at different scales.
Asunto(s)
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Mapas de Interacción de Proteínas/genética , Genes Relacionados con las Neoplasias , Humanos , Mutación , Mapeo de Interacción de Proteínas/métodosRESUMEN
The mapping of SARS-CoV-2 human protein-protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host-virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Reposicionamiento de Medicamentos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , COVID-19 , Ciclo Celular , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/fisiopatología , Daño del ADN , Epigenómica , Humanos , Proteínas de Neoplasias/efectos de los fármacos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/fisiopatología , Pandemias , Neumonía Viral/genética , Neumonía Viral/metabolismo , Neumonía Viral/fisiopatología , Biosíntesis de Proteínas , SARS-CoV-2RESUMEN
The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.
Asunto(s)
Ribonucleoproteínas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Rotación , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación/genética , Dominios Proteicos , Pliegue de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Subunidades Ribosómicas Grandes/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
Asunto(s)
COVID-19/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Interacciones Microbiota-Huesped , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Mapas de Interacción de Proteínas , SARS-CoV-2/metabolismo , Síndrome Respiratorio Agudo Grave/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Secuencia Conservada , Proteínas de la Nucleocápside de Coronavirus/genética , Microscopía por Crioelectrón , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformación ProteicaRESUMEN
An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.
RESUMEN
The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies.