Host Substrate Profiling of SARS-CoV-2 Virus Protease

项目摘要 严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是导致病毒性肺炎爆发的原因 2019冠状病毒病（COVID-19）导致广泛的发病率和死亡率。主要蛋白酶（Mpro） 由SARS-CoV-2编码的蛋白质加工病毒多聚蛋白并促进病毒复制。一旦启动， 酶可以通过切割宿主蛋白质来逃避宿主的先天免疫应答。的宿主蛋白质底物 SARS-CoV-2 Mpro尚未得到很好的表征。直接检测宿主蛋白的主要挑战 目标是其低丰度和技术限制与目前的方法的特异性和灵敏度。 因此，该提议旨在开发一种称为N-末端组学的更特异和敏感的方法， 发现Mpro在SARS CoV-2感染过程中产生的独特蛋白片段。具体目标 1）开发和验证新的探针来鉴定由蛋白酶产生的蛋白酶片段， 细胞裂解液中SARS-CoV-2 Mpro和2）鉴定和定量SARS-CoV-2 Mpro的内源性底物 感染的哺乳动物细胞中。拟议研究的理由是，它的成功将促进更大的 了解SARS-CoV-2改变了哪些宿主途径，从而导致严重疾病。 此外，MPro的宿主靶标可用于开发治疗方案，以治疗COVID-19的严重病例。 这项研究的预期结果是，我们的方法将更敏感，更容易适应 宿主底物分析任何病原性蛋白酶，它将有助于绘制细胞途径， 被病原体劫持以入侵宿主系统。

PROJECT SUMMARY Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the viral pneumonia outbreak of coronavirus disease 2019 (COVID-19) causing widespread morbidity and mortality. Main protease (Mpro) encoded by SARS-CoV-2 processes the viral polyproteins and facilitates viral replication. Once activated, this enzyme can evade the host innate immune responses by cleaving host proteins. The host protein substrates of SARS-CoV-2 Mpro are not well characterized. The major challenges with the direct detection of host protein targets is their low abundance and technical limitations with the specificity and sensitivity of the current methods. Thus, this proposal is designed to develop a more specific and sensitive approach termed N-terminomics to discover unique protein fragments that are generated by Mpro during SARS CoV-2 infection. The Specific Aims of this proposal are: 1) Development and validation of novel probes to identify protease fragments generated by SARS-CoV-2 Mpro in cell lysate and 2) Identify and quantify the endogenous substrates of SARS-CoV-2 Mpro in infected mammalian cells. The rationale for the proposed research is that its success would facilitate a greater understanding of which host pathways are altered by this SARS-CoV-2 to cause a severe disease condition. Moreover, host targets of MPro can be used to develop treatment regimens to treat severe cases if COVID-19. The expected outcome of this research is that our approach will be more sensitive and readily adaptable for the host substrate profiling of any pathogenic proteases and it will aid in mapping cellular pathways which are hijacked by pathogens to invade the host system.