Zhang Yaotian, Stöppelkamp Ida, Fernandez-Pernas Pablo, Allram Melanie, Charman Matthew, Magalhaes Alexandre P, Piedavent-Salomon Melanie, Sommer Gregor, Sung Yu-Chieh, Meyer Katrina, Grams Nicholas, Halko Edwin, Dongre Shivali, Meierhofer David, Malszycki Michal, Ilik Ibrahim A, Aktas Tugce, Kraushar Matthew L, Vastenhouw Nadine, Weitzman Matthew D, Grebien Florian, Niskanen Henri, Hnisz Denes
Max Planck Institute for Molecular Genetics, Berlin, Germany.
Institute of Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany.
Nature. 2025 Jun 4. doi: 10.1038/s41586-025-09141-5.
Biomolecular condensates are thought to create subcellular microenvironments that have different physicochemical properties compared with their surrounding nucleoplasm or cytoplasm. However, probing the microenvironments of condensates and their relationship to biological function is a major challenge because tools to selectively manipulate specific condensates in living cells are limited. Here, we develop a non-natural micropeptide (that is, the killswitch) and a nanobody-based recruitment system as a universal approach to probe endogenous condensates, and demonstrate direct links between condensate microenvironments and function in cells. The killswitch is a hydrophobic, aromatic-rich sequence with the ability to self-associate, and has no homology to human proteins. When recruited to endogenous and disease-specific condensates in human cells, the killswitch immobilized condensate-forming proteins, leading to both predicted and unexpected effects. Targeting the killswitch to the nucleolar protein NPM1 altered nucleolar composition and reduced the mobility of a ribosomal protein in nucleoli. Targeting the killswitch to fusion oncoprotein condensates altered condensate compositions and inhibited the proliferation of condensate-driven leukaemia cells. In adenoviral nuclear condensates, the killswitch inhibited partitioning of capsid proteins into condensates and suppressed viral particle assembly. The results suggest that the microenvironment within cellular condensates has an essential contribution to non-stoichiometric enrichment and mobility of effector proteins. The killswitch is a widely applicable tool to alter the material properties of endogenous condensates and, as a consequence, to probe functions of condensates linked to diverse physiological and pathological processes.
生物分子凝聚物被认为可创造出与周围核质或细胞质相比具有不同物理化学性质的亚细胞微环境。然而，探究凝聚物的微环境及其与生物学功能的关系是一项重大挑战，因为在活细胞中选择性操纵特定凝聚物的工具有限。在此，我们开发了一种非天然微肽（即“杀伤开关”）和一种基于纳米抗体的招募系统，作为探测内源性凝聚物的通用方法，并证明了凝聚物微环境与细胞功能之间的直接联系。“杀伤开关”是一种具有自我缔合能力的富含疏水芳香族的序列，与人类蛋白质无同源性。当被招募到人类细胞中的内源性和疾病特异性凝聚物时，“杀伤开关”会固定形成凝聚物的蛋白质，从而产生预期和意外的效果。将“杀伤开关”靶向核仁蛋白NPM1会改变核仁组成，并降低核糖体蛋白在核仁中的流动性。将“杀伤开关”靶向融合癌蛋白凝聚物会改变凝聚物组成，并抑制凝聚物驱动的白血病细胞的增殖。在腺病毒核凝聚物中，“杀伤开关”抑制衣壳蛋白向凝聚物中的分配，并抑制病毒颗粒组装。结果表明，细胞凝聚物中的微环境对效应蛋白的非化学计量富集和流动性有重要贡献。“杀伤开关”是一种广泛适用的工具，可改变内源性凝聚物的物质特性，从而探测与各种生理和病理过程相关的凝聚物功能。
