School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan.
J Pept Sci. 2024 Feb;30(2):e3536. doi: 10.1002/psc.3536. Epub 2023 Aug 14.
Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.
蛋白质聚集是各种细胞过程中普遍存在的事件。蛋白质的自我缔合会触发下游蛋白质的募集，从而调节细胞信号转导。为了详细研究这些相互作用，需要使用化学生物学工具来鉴定募集到特定组装体的蛋白质。在这里，我们利用一种结合了基于 Y15 的细胞内超分子构建和邻近标记方法的人工颗粒（IPRAG）平台来鉴定蛋白质募集。我们使用 Nck1 作为靶标诱饵蛋白来验证 IPRAG 工具。我们构建了 Nck1 结合颗粒，用生物素标记募集的蛋白质，然后通过 LC-MS/MS 进行分析。结果，我们成功地鉴定了与 Nck1 直接或间接相互作用的蛋白质。
