RESUMO
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.
Assuntos
COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mapas de Interação de Proteínas , SARS-CoV-2/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Sequência Conservada , Proteínas do Nucleocapsídeo de Coronavírus/genética , Microscopia Crioeletrônica , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformação ProteicaRESUMO
Essential characteristics of cellular signaling networks include a complex interconnected architecture and temporal dynamics of protein activity. The latter can be monitored by Förster resonance energy transfer (FRET) biosensors at a single-live-cell level with high temporal resolution. However, these experiments are typically limited to the use of a couple of FRET biosensors. Here, we describe a FRET-based multi-parameter imaging platform (FMIP) that allows simultaneous high-throughput monitoring of multiple signaling pathways. We apply FMIP to monitor the crosstalk between epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor signaling, signaling perturbations caused by pathophysiologically relevant EGFR mutations, and the effects of a clinically important MEK inhibitor (selumetinib) on the EGFR network. We expect that in the future the platform will be applied to develop comprehensive models of signaling networks and will help to investigate the mechanism of action as well as side effects of therapeutic treatments.
