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.

中文翻译：

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基于HaloTag的多色荧光传感器使用溶剂变色和基于分子转子的荧光团可视化和量化活细胞中的蛋白质组应力。

蛋白质稳态或蛋白稳态对于细胞适应性和生存能力至关重要。许多环境因素都会损害蛋白质稳态，引起整体蛋白质组压力，并导致疾病。蛋白质组应力传感器是剖析细胞应力机制并找到可缓解这些疾病的疗法的强大工具。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），以可视化和量化活细胞中蛋白质组的压力。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环境中暴露于压力条件下形成可溶性低聚物或不溶性聚集体时，它们会开启荧光。此外，AgHalo探针可以与市售的始终发光的HaloTag配体结合使用，以实现双色成像，从而可以在细胞遭受压力条件之前和之后直接可视化AgHalo传感器。最后，可以通过首先形成AgHalo共轭物，然后在任何所需的时间点施加或消除压力，来进行脉冲追踪实验来识别活细胞中细胞蛋白质组的变化。总而言之，AgHalo传感器可用于可视化和量化活细胞中的蛋白质组应力，这是使用以前的始终荧光方法很难完成的任务。该传感器应适合评估各种外源压力下的细胞蛋白水解，包括化学毒素，药物和环境因素。