Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
Structural Genomics Consortium, Toronto, ON, Canada.
Methods Mol Biol. 2021;2318:45-67. doi: 10.1007/978-1-0716-1476-1_4.
By identifying MYC protein-protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein-protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo-in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.
通过鉴定 MYC 蛋白-蛋白相互作用物，我们旨在深入了解 MYC 作为基因转录调控因子和强效癌蛋白的作用机制。然后，可以利用这些信息来设计破坏关键 MYC 蛋白-蛋白相互作用的策略，以抑制 MYC 驱动的肿瘤发生。在本章中，我们讨论了四种鉴定和验证 MYC 相互作用伙伴的技术。首先，我们重点介绍了 BioID，这是一种强大的发现方法，用于鉴定活细胞中高可信度的近邻相互作用物。我们还讨论了用于鉴定 BioID 衍生的 MYC 近端复合物的生物信息学优先级策略。接下来，我们讨论了如何使用体内-体外下拉测定法和邻近连接测定法 (PLA) 等技术验证蛋白质相互作用。最后，我们概述了生物层干涉法 (BLI)，这是一种用于体外直接鉴定两种蛋白质之间相互作用的定量方法。总之，我们强调了每种测定方法的原理，并提供了进行这些实验所需的方法，以及将其改编为研究其他感兴趣的蛋白质相互作用物的方法。
