The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasomein vitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substratein vitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomal deubiquitinase Rpn11 (POH1/PSMD14). ETPs also inhibited other JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases such as Csn5 and AMSH. An improved ETP with fewer non-specific effects, SOP11, stabilized a subset of proteasome substrates in cells, induced the unfolded protein response, and led to cell death. SOP11 represents a class of Rpn11 inhibitor and provides an alternative route to develop proteasome inhibitors.

中文翻译：

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Epidithiodiketopiperazines通过靶向蛋白酶体去泛素酶Rpn11抑制蛋白质降解。

26S蛋白酶体是分解真核生物中胞质和核蛋白的主要蛋白水解机器。由于缺乏合适的检测方法，因此很难通过常规方法和定量方法检测26S蛋白酶体在体外对蛋白质的分解。在本研究中，我们开发了一种检测蛋白酶体介导的蛋白质降解的方法。使用该测定法，我们发现表二硫代二酮哌嗪（ETPs）在体外阻断了我们模型基质的降解。进一步的特征表明，ETP通过靶向必需的蛋白酶体去泛素化酶Rpn11（POH1 / PSMD14）来抑制蛋白酶体功能。ETP还抑制其他JAMM（JAB1 / MPN / Mov34金属酶）蛋白酶，例如Csn5和AMSH。具有较少非特异性作用的改良ETP SOP11可稳定细胞中蛋白酶体底物的一部分，诱导展开的蛋白质反应，并导致细胞死亡。SOP11代表一类Rpn11抑制剂，并提供了开发蛋白酶体抑制剂的替代途径。