Department of Medicinal Chemistry and Molecular Pharmacology , Purdue University , 575 West Stadium Avenue , West Lafayette , Indiana 47907 , United States.
J Am Chem Soc. 2019 Apr 3;141(13):5252-5260. doi: 10.1021/jacs.8b12873. Epub 2019 Mar 20.
Activity-based probes have greatly improved our understanding of the intrinsic roles and expression levels of various proteins within cells. To be useful in live cells, probes must be cell permeable and provide a read-out that can be measured without disrupting the cells or the activity of the target. Unfortunately, probes for the various forms of the proteasome that can be utilized in intact cells are limited; commercially available probes are most effectively used with purified protein or cell lysate. The proteasome, both the 26S and various isoforms of the 20S CP, is an important target with reported roles in cancer, autoimmune disorders, and neurodegenerative diseases. Here, we present the development of a selective probe for the immunoproteasome, a specialized isoform of the 20S proteasome, that becomes expressed in cells that encounter an inflammatory signal. Using a one-bead, one-compound library of small peptides, we discovered a trimer sequence efficiently cleaved by the immunoproteasome with significant selectivity over the standard proteasome. Upon conjugating this sequence to rhodamine 110 and a peptoid, we generated a probe with a considerable improvement in sensitivity compared to that of current aminomethylcoumarin-based proteasome probes. Importantly, our probe was capable of labeling immunoproteasome-expressing cells while maintaining its selectivity over other cellular proteases in live cell cultures. We anticipate this probe to find wide utility for those that wish to study the immunoproteasome's activity in a variety of cell lines and to be used as a reporter to discover small molecules that can perturb the activity of this proteasome isoform.
活性探针极大地提高了我们对细胞内各种蛋白质固有作用和表达水平的理解。为了在活细胞中有用，探针必须能够穿透细胞，并提供一种可以测量的读数，而不会干扰细胞或靶标活性。不幸的是，可用于完整细胞的各种形式蛋白酶体的探针是有限的；市售探针最有效地用于纯化蛋白或细胞裂解物。蛋白酶体，包括 26S 和各种 20S CP 同工型，是一个重要的靶点，据报道在癌症、自身免疫性疾病和神经退行性疾病中发挥作用。在这里，我们提出了一种免疫蛋白酶体的选择性探针的开发，这是 20S 蛋白酶体的一种特殊同工型，在遇到炎症信号的细胞中表达。使用一个小肽的一个珠子、一个化合物库，我们发现了一个三聚体序列，该序列被免疫蛋白酶体有效地切割，与标准蛋白酶体相比具有显著的选择性。在将该序列与罗丹明 110 和肽缩合后，我们生成了一种探针，与目前基于氨甲基香豆素的蛋白酶体探针相比，其灵敏度有了相当大的提高。重要的是，我们的探针能够标记表达免疫蛋白酶体的细胞，同时保持其对活细胞培养物中其他细胞蛋白酶的选择性。我们预计该探针将在各种细胞系中研究免疫蛋白酶体的活性方面得到广泛应用，并用作发现能够扰乱这种蛋白酶体同工型活性的小分子的报告器。
