RESUMO
Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Camundongos , Linhagem CelularRESUMO
Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.
Assuntos
Chlamydomonas reinhardtii , Cílios , Glicoproteínas , Cílios/química , Glicoproteínas/química , Glicosilação , Hidroxiprolina/química , Plantas/metabolismo , Chlamydomonas reinhardtii/químicaRESUMO
We performed comprehensive proteogenomic characterization of small cell lung cancer (SCLC) using paired tumors and adjacent lung tissues from 112 treatment-naive patients who underwent surgical resection. Integrated multi-omics analysis illustrated cancer biology downstream of genetic aberrations and highlighted oncogenic roles of FAT1 mutation, RB1 deletion, and chromosome 5q loss. Two prognostic biomarkers, HMGB3 and CASP10, were identified. Overexpression of HMGB3 promoted SCLC cell migration via transcriptional regulation of cell junction-related genes. Immune landscape characterization revealed an association between ZFHX3 mutation and high immune infiltration and underscored a potential immunosuppressive role of elevated DNA damage response activity via inhibition of the cGAS-STING pathway. Multi-omics clustering identified four subtypes with subtype-specific therapeutic vulnerabilities. Cell line and patient-derived xenograft-based drug tests validated the specific therapeutic responses predicted by multi-omics subtyping. This study provides a valuable resource as well as insights to better understand SCLC biology and improve clinical practice.
Assuntos
Neoplasias Pulmonares , Proteogenômica , Carcinoma de Pequenas Células do Pulmão , Humanos , Linhagem Celular , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/química , Carcinoma de Pequenas Células do Pulmão/genética , Xenoenxertos , Biomarcadores Tumorais/análiseRESUMO
Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.
Assuntos
Glicoproteínas , Simulação de Dinâmica Molecular , Humanos , Microscopia Crioeletrônica , Glicoproteínas/química , Glicosilação , Polissacarídeos/químicaRESUMO
The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.
RESUMO
Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.
Assuntos
Neoplasias , Processamento de Proteína Pós-Traducional , Proteômica , Humanos , Acetilação , Histonas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Proteômica/métodosRESUMO
To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.
Assuntos
Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Proteogenômica , Feminino , Humanos , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genéticaRESUMO
Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
Assuntos
Glucagon , Receptores de Glucagon , Membrana Celular/metabolismo , Glucagon/metabolismo , Receptores de Glucagon/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/metabolismoRESUMO
In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies and methods allow accurate identification, transcriptome-wide mapping, and functional characterization of a growing number of RNA modifications, providing important insights into the biology of these marks. Most of the methods and approaches were developed for studying m6A, the most prevalent internal mRNA modification. However, unique properties of other RNA modifications stimulated the development of additional approaches. In this technical primer, we will discuss the available tools and approaches for detecting and studying different RNA modifications.
Assuntos
Processamento Pós-Transcricional do RNA , RNA , Epigênese Genética , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
Assuntos
Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Mapas de Interação de Proteínas , Sinapses/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilação , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Progressão da Doença , Metabolismo Energético , Demência Frontotemporal/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Degeneração Neural/patologia , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Proteômica , Índice de Gravidade de Doença , Frações Subcelulares/metabolismo , Tauopatias/genética , Proteínas tau/químicaRESUMO
Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.
Assuntos
Doença de Alzheimer , Proteoma , Camundongos , Humanos , Animais , Proteoma/análise , Proteômica/métodos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Espectrometria de Massas , Placa AmiloideRESUMO
HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Vírion/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Sequência de Aminoácidos , Dissulfetos/farmacologia , Epitopos/química , Células HEK293 , Proteína gp41 do Envelope de HIV/química , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Moleculares , Testes de Neutralização , Peptídeos/química , Polissacarídeos/química , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.
Assuntos
Especificidade de Órgãos , Mapeamento de Interação de Proteínas , Animais , Marcação por Isótopo , Masculino , Mamíferos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Aminoacil-tRNA Sintetases/metabolismo , Antituberculosos/farmacologia , Teorema de Bayes , Evolução Biológica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.
Assuntos
Proteínas de Escherichia coli/metabolismo , Imageamento Tridimensional , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Espectrometria de Massas , Simulação de Dinâmica Molecular , Pressão Osmótica , Fosforilação , Proteólise , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estresse FisiológicoRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteogenômica , Adenocarcinoma de Pulmão/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas de Fusão Oncogênica , Fenótipo , Fosfoproteínas/metabolismo , Proteoma/metabolismoRESUMO
Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.
Assuntos
Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas/fisiologia , Espectrometria de Massas/métodos , Plantas/genética , Plantas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodosRESUMO
The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this "proteogenomics" approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases. Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/patologia , Terapia de Alvo Molecular , Proteogenômica , Desaminases APOBEC/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Estudos de Coortes , Dano ao DNA , Reparo do DNA , Feminino , Humanos , Imunoterapia , Metabolômica , Pessoa de Meia-Idade , Mutagênese/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Receptor ErbB-2/metabolismo , Proteína do Retinoblastoma/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.
Assuntos
Vírus Gigantes/genética , Vírus Gigantes/metabolismo , Vírus Gigantes/fisiologia , Capsídeo/metabolismo , Vírus de DNA/genética , Genoma Viral/genética , Proteômica/métodos , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Viroses/genética , Vírus/genéticaRESUMO
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.