Warren Julia, Wang Jian, Dhoro Francis, Chao Bo, Reddy Ashok, Petrie Stefanie Kaech, David Larry L, Xiao Xiangshu, Li Bingbing X
Program in Chemical Biology, Department of Chemical Physiology and Biochemistry, Proteomics Shared Resources, Knight Cancer Institute, Department of Neurology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, Oregon 97239, United States.
JACS Au. 2025 Jan 14;5(1):302-319. doi: 10.1021/jacsau.4c00988. eCollection 2025 Jan 27.
Proteins regulate biological functions through the formation of distinct protein complexes. Identification and characterization of these protein-protein interactions are critical to deciphering their mechanism of action. Different antibody-based or cross-linking-based methods have been developed to identify the protein-protein interactions. However, these methods require genetic engineering or other means to disrupt the native environments. To circumvent this limitation, we introduce here SMAP3-ID (small-molecule-assisted identification of protein-protein interactions through proximity) method to identify protein-protein interactions in native cellular environment. This method combines a selective ligand for binding to a protein of interest for photo-cross-linking, a live-cell-compatible bioorthogonal click reaction with a trifunctional chemical probe, and a final photo-cross-linking reaction to covalently capture the interacting proteins. Using the SMAP3-ID method and nuclear lamins as an example, we identified numerous lamin interactors in native cells. Significantly, we identified a number of mitochondrial enzymes as novel lamin A (LA) interactors. The interactions between mitochondrial enzymes and LA were further validated, which provides mechanistic insights underlying the metabolic alterations caused by mutations in LA. Furthermore, our previously described small-molecule ligand for LA, , also induced changes in mitochondrial activity and cellular bioenergetic organization. We conclude that SMAP3-ID is a potentially powerful and generalizable method to identify protein-protein interactions in the native cellular environment.
蛋白质通过形成独特的蛋白质复合物来调节生物学功能。识别和表征这些蛋白质-蛋白质相互作用对于解读其作用机制至关重要。已经开发出了不同的基于抗体或基于交联的方法来识别蛋白质-蛋白质相互作用。然而，这些方法需要基因工程或其他手段来破坏天然环境。为了克服这一限制，我们在此引入SMAP3-ID（通过邻近性小分子辅助识别蛋白质-蛋白质相互作用）方法，以在天然细胞环境中识别蛋白质-蛋白质相互作用。该方法将用于与感兴趣蛋白质结合以进行光交联的选择性配体、与三功能化学探针的活细胞兼容生物正交点击反应以及最终的光交联反应结合起来，以共价捕获相互作用的蛋白质。以SMAP3-ID方法和核纤层蛋白为例，我们在天然细胞中鉴定出了众多核纤层蛋白相互作用蛋白。值得注意的是，我们鉴定出了一些线粒体酶作为新型核纤层蛋白A（LA）相互作用蛋白。线粒体酶与LA之间的相互作用得到了进一步验证，这为LA突变引起的代谢改变提供了潜在的作用机制。此外，我们之前描述的用于LA的小分子配体也会诱导线粒体活性和细胞生物能量组织的变化。我们得出结论，SMAP3-ID是一种在天然细胞环境中识别蛋白质-蛋白质相互作用的潜在强大且通用的方法。
