The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity toward the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity.

中文翻译：

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带有非天然氨基酸的荧光探针实时监测蛋白酶体活性。

蛋白酶体是一种必需的蛋白质复合物，当其失调时，会导致真核细胞发生各种疾病。因此，了解蛋白酶体的酶活性以及可以改变它的因素对于阐明其在这些疾病中的作用至关重要。这可以通过使用基于活性的荧光底物探针有效地完成，其中有许多商业上可用的靶向蛋白酶体 20S CP 中的单个蛋白酶样亚基。不幸的是，这些探针没有显示出适合它们用于基于活细胞的测定的特性。在这里介绍的工作中，我们开发了一组探针，与其他细胞蛋白酶相比，这些探针显示出改进的荧光特性和对蛋白酶体的选择性。通过加入非天然氨基酸，我们发现了可用于各种应用的探针，包括监测活细胞中蛋白酶体的小分子刺激剂的作用，以及比较不同癌细胞类型的相对蛋白酶体活性。在未来的研究中，我们期望这里介绍的荧光探针将作为工具来支持发现和表征蛋白酶体活性的小分子调节剂。