20S 蛋白酶体是一种多聚体蛋白酶复合物，对于细胞内的蛋白质稳态至关重要。小分子蛋白酶体抑制剂是已批准用于治疗各种癌症的药物，并且作为抗寄生虫药正在临床上取得进展。尽管研究 20S 蛋白酶体的工具和技术已经取得了进步，但只有一种商业探针可用于对蛋白酶体活性进行成像。该探针由荧光标记的肽基乙烯基砜组成，可与一个或多个催化蛋白酶体亚基结合。在这里，我们合成了两种活性位点定向环氧酮探针 LJL-1 和 LJL-2，它们分别基于抗癌药物卡非佐米和硼替佐米的肽基骨架。每个探针通过点击化学与包含 5-羧基四甲基罗丹明 (TAMRA) 和生物素的双功能基团缀合，分别从杜氏利什曼原虫和阴道毛滴虫这两种真核病原体的蛋白质提取物中可视化和富集 20S 蛋白酶体。根据物种的不同，每个探针通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）产生不同的亚基结合谱，生物素标签能够富集结合的亚基，然后通过蛋白质组学正式鉴定这些亚基。还注意到β亚基电泳迁移顺序的物种差异。最后，两种探针都与 20S 亚基发生特异性反应，这与与半胱氨酸蛋白酶发生交叉反应的市售乙烯基砜探针不同。 LJL-1 和 LJL-2 应在病原体蛋白酶体的鉴定和表征中具有通用性，并可作为评估新型抗寄生虫蛋白酶体抑制剂的特异性和结合机制的试剂。© 2024 作者。由美国化学会出版。

The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the β subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.© 2024 The Authors. Published by American Chemical Society.