Hazegh Nikroo Arjan, Altenburg Wiggert J, van Veldhuisen Thijs W, Brunsveld Luc, van Hest Jan C M
Laboratory of Bio-Organic Chemistry, Department of Biomedical Engineering, and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven, 5600 MB, The Netherlands.
Laboratory of Chemical Biology, Department of Biomedical Engineering, and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven, 5600 MB, The Netherlands.
Adv Biol (Weinh). 2025 May;9(5):e2400353. doi: 10.1002/adbi.202400353. Epub 2024 Sep 27.
The regulation of protein uptake and secretion by cells is paramount for intercellular signaling and complex multicellular behavior. Mimicking protein-mediated communication in artificial cells holds great promise to elucidate the underlying working principles, but remains challenging without the stimulus-responsive regulatory machinery of living cells. Therefore, systems to precisely control when and where protein release occurs should be incorporated in artificial cells. Here, a light-activatable TEV protease (LaTEV) is presented that enables spatiotemporal control over protein release from a coacervate-based artificial cell platform. Due to the presence of Ni-nitrilotriacetic acid moieties within the coacervates, His-tagged proteins are effectively sequestered into the coacervates. LaTEV is first photocaged, effectively blocking its activity. Upon activation by irradiation with 365 nm light, LaTEV cleaves the His-tags from sequestered cargo proteins, resulting in their release. The successful blocking and activation of LaTEV provides control over protein release rate and triggerable protein release from specific coacervates via selective irradiation. Furthermore, light-activated directional transfer of proteins between two artificial cell populations is demonstrated. Overall, this system opens up avenues to engineer light-responsive protein-mediated communication in artificial cell context, which can advance the probing of intercellular signaling and the development of protein delivery platforms.
细胞对蛋白质摄取和分泌的调节对于细胞间信号传导和复杂的多细胞行为至关重要。在人工细胞中模拟蛋白质介导的通讯有望阐明其潜在的工作原理，但在缺乏活细胞的刺激响应调节机制的情况下仍然具有挑战性。因此，应将能够精确控制蛋白质释放时间和位置的系统整合到人工细胞中。在此，我们展示了一种光激活的烟草蚀纹病毒蛋白酶（LaTEV），它能够对基于凝聚层的人工细胞平台中的蛋白质释放进行时空控制。由于凝聚层中存在镍-次氮基三乙酸部分，带有His标签的蛋白质被有效地隔离到凝聚层中。LaTEV首先被光笼化，有效地阻断其活性。在用365 nm光照射激活后，LaTEV从被隔离的货物蛋白上切割His标签，导致它们释放。LaTEV的成功阻断和激活通过选择性照射提供了对蛋白质释放速率的控制以及从特定凝聚层中触发蛋白质释放的能力。此外，还展示了蛋白质在两个人工细胞群体之间的光激活定向转移。总体而言，该系统为在人工细胞环境中设计光响应性蛋白质介导的通讯开辟了途径，这可以推动对细胞间信号传导的探索以及蛋白质递送平台的发展。
