Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
J Am Chem Soc. 2021 May 5;143(17):6434-6446. doi: 10.1021/jacs.0c12375. Epub 2021 Apr 23.
Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.
蛋白质被分隔在生物分子凝聚物或无膜细胞器中，这些凝聚物通过相分离形成，这是调节细胞过程的一个新兴原则。创建能够按需容纳特定细胞内蛋白质的合成凝聚物，将在化学生物学、细胞工程和合成生物学中有各种应用。在这里，我们报告了合成蛋白质凝聚物的构建，这些凝聚物能够响应小分子或光在活哺乳动物细胞中招募和/或释放感兴趣的蛋白质。通过串联融合两种寡聚蛋白的模块化组合，这些蛋白在细胞中形成相分离的合成蛋白质凝聚物，再加上化学诱导的二聚化工具，我们首先创建了一种化学生物学蛋白质凝聚物系统，通过添加小分子二聚化剂，可以将靶蛋白从细胞质快速招募到凝聚物中。接下来，我们将蛋白招募凝聚物系统与工程化的近邻依赖性蛋白酶偶联，得到了第二个蛋白质凝聚物系统，其中先前在凝聚物内表达的靶蛋白可以通过小分子触发的蛋白酶招募释放到细胞质中。此外，还成功构建了一种光遗传学凝聚物系统，该系统可以使用光以可重复的方式可逆地释放和隔离蛋白质活性。这些凝聚物系统可用于控制蛋白质活性和细胞过程，例如在几分钟的时间尺度内控制细胞膜皱襞和 ERK 信号。这项原理验证工作为化学生物学和光遗传学控制哺乳动物细胞中蛋白质活性提供了一个新平台，代表着朝着具有各种功能的基于合成蛋白质凝聚物的软材料的定制工程迈出了一步，可用于生物和生物医学应用。
