A HaloTag-Based Multicolor Fluorogenic Sensor Visualizes and Quantifies Proteome Stress in Live Cells Using Solvatochromic and Molecular Rotor-Based Fluorophores
A HaloTag-Based Multicolor Fluorogenic Sensor Visualizes and Quantifies Proteome Stress in Live Cells Using Solvatochromic and Molecular Rotor-Based Fluorophores
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DOI：
10.1021/acs.biochem.8b00135
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发表时间：
2018-08-07
期刊：
影响因子：
2.9
通讯作者：
Zhang, Xin
中科院分区：
文献类型：
--
作者：
Liu, Yu;Miao, Kun;Zhang, Xin
蛋白质稳态或蛋白质稳态对于细胞健康和活力至关重要。许多环境因素会损害蛋白质稳态，诱发整体蛋白质组应激，并导致疾病。蛋白质组应激传感器是剖析细胞应激机制并寻找改善这些疾病的治疗方法的强大工具。在这项工作中，我们提出了一种基于 HaloTag 的多色传感器（名为 AgHalo），用于可视化和量化活细胞中的蛋白质组应力。当前的 AgHalo 传感器配备了三个荧光探针，当传感器在体外和纤维素中暴露于应激条件下形成可溶性低聚物或不溶性聚集体时，这些探针会打开荧光。此外，AgHalo 探针可以与市售的始终荧光的 HaloTag 配体结合使用，以实现双色成像，从而可以在细胞受到应激条件之前和之后直接可视化 AgHalo 传感器。最后，可以进行脉冲追踪实验，通过首先形成 AgHalo 缀合物，然后在任何所需时间点施加或消除应力，来辨别活细胞中细胞蛋白质组的变化。总之，AgHalo 传感器可用于可视化和量化活细胞中的蛋白质组应激，这是使用以前的始终荧光方法难以完成的任务。该传感器应该适合评估各种外源应激下的细胞蛋白质稳态，包括化学毒素、药物和环境因素。
Protein homeostasis, or proteostasis, is essential for cellular fitness and viability. Many environmental factors compromise proteostasis, induce global proteome stress, and cause diseases. The proteome stress sensor is a powerful tool for dissecting the mechanism of cellular stress and finding therapeutics that ameliorate these diseases. In this work, we present a multicolor HaloTag-based sensor (named AgHalo) to visualize and quantify proteome stresses in live cells. The current AgHalo sensor is equipped with three fluorogenic probes that turn on fluorescence when the sensor forms either soluble oligomers or insoluble aggregates upon exposure to stress conditions, both in vitro and in cellulo. In addition, AgHalo probes can be combined with commercially available always-fluorescent HaloTag ligands to enable two-color imaging, allowing for direct visualization of the AgHalo sensor both before and after cells are subjected to stress conditions. Finally, pulse-chase experiments can be performed to discern changes in the cellular proteome in live cells by first forming the AgHalo conjugate and then either applying or removing stress at any desired time point. In summary, the AgHalo sensor can be used to visualize and quantify proteome stress in live cells, a task that is difficult to accomplish using previous always-fluorescent methods. This sensor should be suited to evaluating cellular proteostasis under various exogenous stresses, including chemical toxins, drugs, and environmental factors.