基于相分离凝聚体的生物分子互作检测方法和高通量药物筛选平台
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2019YFA0508403-003。相分离凝聚体的组分分为Scaffold和Client两类，Scaffold能通过多价互作驱动相分离，是维持凝聚体所必需的；Client通过结合Scaffold被特异性的招募到凝聚体中。利用Client的招募特性，我们开发了一个基于相分离凝聚体的生物分子互作检测方法CEBIT，可检测各种生物分子互作，包括蛋白-蛋白互作、蛋白的翻译后修饰以及DNA和RNA的甲基化修饰。此外，还可以检测蛋白质修饰酶的活性。在此基础上，通过高通量筛选，发现各种生物分子互作及酶的抑制剂。该方法为我们研究各种相分离凝聚体内复杂的分子互作，开发靶向致病性凝聚体的药物提供了重要的技术平台。 此外我们开发了一种新的在活细胞中研究蛋白-蛋白相互作用的方法CoPIC。这种方法利用相分离凝聚体，在细胞内特定的相互作用可以使蛋白质被招募到这些凝聚体中。通过利用CoPIC筛选，我们绘制了SARS-CoV-2与复制/转录复合体相关的病毒蛋白之间的相互作用网络。我们共鉴定出14种蛋白之间的47种二元相互作用，这些蛋白控制着冠状病毒的复制、间断转录和翻译。我们发现了由这些组分组成的广泛的三元复合体，这些复合体暗示了潜在的更高级复合体的存在。总的来说，我们的结果为理解SARS-CoV-2的生物学特性和开发治疗COVID-19的干预措施提供了机会，构建了基于相分离凝聚体的生物分子互作检测方法和高通量药物筛选平台，还检测了组蛋白H3K9的甲基化修饰、DNA和RNA的甲基化修饰以及H3K9的甲基化酶SUV39H1。进一步，我们以p53-MDM2和SUV39H1为靶点，分别做了高通量药物筛选并验证；开发了一种新的在活细胞中研究蛋白-蛋白相互作用的方法CoPIC，绘制了与复制/转录复合体相关的病毒蛋白之间的相互作用网络，开发调控这些分子互作网络的靶向药物，干预异常相分离导致的神经疾病。
Project No. 2019YFA0508403-003. The components of phase-separated condensates are categorized into two classes: Scaffold and Client. Scaffolds drive phase separation via multivalent interactions and are indispensable for maintaining condensates; Clients are specifically recruited into condensates through binding to Scaffolds. Leveraging the recruitment characteristics of Clients, we developed a biomolecular interaction detection assay CEBIT based on phase-separated condensates. This assay enables detection of diverse biomolecular interactions, including protein-protein interactions, post-translational modifications of proteins, and methylation modifications of DNA and RNA. Additionally, it can also measure the activity of protein-modifying enzymes. On this basis, we conducted high-throughput screening to discover inhibitors of various biomolecular interactions and enzymes. This method provides a critical technical platform for investigating complex molecular interactions within diverse phase-separated condensates and developing drugs targeting pathogenic condensates. Furthermore, we developed a novel live-cell assay CoPIC for studying protein-protein interactions. This assay utilizes phase-separated condensates, where specific intracellular interactions lead to the recruitment of target proteins into these condensates. Using CoPIC screening, we mapped the interaction network between SARS-CoV-2 viral proteins associated with the replication/transcription complex. We identified a total of 47 binary interactions among 14 proteins that govern coronavirus replication, discontinuous transcription, and translation. We discovered extensive ternary complexes formed by these components, which imply the presence of potential higher-order complexes. Overall, our findings offer opportunities to understand the biological properties of SARS-CoV-2 and develop COVID-19 intervention strategies. We established biomolecular interaction detection assays and high-throughput drug screening platforms based on phase-separated condensates, and detected methylation modifications of histone H3K9, DNA and RNA, as well as the H3K9 methyltransferase SUV39H1. Furthermore, we performed and validated high-throughput drug screens targeting p53-MDM2 and SUV39H1, respectively. We developed a novel live-cell protein-protein interaction assay CoPIC, mapped the interaction network between viral proteins associated with the replication/transcription complex, developed targeted drugs that regulate these molecular interaction networks, and intervened in neurological diseases caused by abnormal phase separation.
