Pan Chenmengxiao Roderick, Knutson Steve D, Huth Sean W, MacMillan David W C
Merck Center for Catalysis at Princeton University, Princeton, NJ, USA.
Department of Chemistry, Princeton University, Princeton, NJ, USA.
Nat Chem Biol. 2025 Apr;21(4):490-500. doi: 10.1038/s41589-024-01721-2. Epub 2024 Aug 30.
Phase-separated condensates are membrane-less intracellular structures comprising dynamic protein interactions that organize essential biological processes. Understanding the composition and dynamics of these organelles advances our knowledge of cellular behaviors and disease pathologies related to granule dysregulation. In this study, we apply microenvironment mapping with a HaloTag-based platform (HaloMap) to characterize intracellular stress granule dynamics in living cells. After validating the robustness and sensitivity of this approach, we then profile the stress granule proteome throughout the formation and disassembly and under pharmacological perturbation. These experiments reveal several ubiquitin-related modulators, including the HECT (homologous to E6AP C terminus) E3 ligases ITCH and NEDD4L, as well as the ubiquitin receptor toll-interacting protein TOLLIP, as key mediators of granule disassembly. In addition, we identify an autophagy-related pathway that promotes granule clearance. Collectively, this work establishes a general photoproximity labeling approach for unraveling intracellular protein interactomes and uncovers previously unexplored regulatory mechanisms of stress granule dynamics.
相分离凝聚物是无膜的细胞内结构，由动态蛋白质相互作用组成，这些相互作用组织着重要的生物学过程。了解这些细胞器的组成和动态变化，有助于我们深入了解与颗粒失调相关的细胞行为和疾病病理。在本研究中，我们应用基于HaloTag的平台（HaloMap）进行微环境映射，以表征活细胞内应激颗粒的动态变化。在验证了该方法的稳健性和敏感性之后，我们对整个应激颗粒蛋白质组在形成、解体过程以及药物扰动下的情况进行了分析。这些实验揭示了几种与泛素相关的调节因子，包括HECT（与E6AP C末端同源）E3连接酶ITCH和NEDD4L，以及泛素受体Toll相互作用蛋白TOLLIP，它们是颗粒解体的关键介质。此外，我们还确定了一条促进颗粒清除的自噬相关途径。总的来说，这项工作建立了一种用于揭示细胞内蛋白质相互作用组的通用光邻近标记方法，并揭示了应激颗粒动态变化中以前未被探索的调节机制。
