hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

中文翻译：

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丢失hRpn13 Pru或UCHL5对泛素化蛋白的蛋白酶体清除和RA190细胞毒性的影响。

hRpn13 / ADRM1通过其蛋白酶体和泛素结合的Pru结构域和DEUBAD结构域将底物募集与蛋白酶体上的去泛素化联系起来，后者结合并激活去泛素化酶（DUB）UCHL5 / Uch37。在这里，我们编辑HCT116大肠癌细胞系以删除hRpn13 Pru的一部分，从而产生表达截短的hRpn13（trRpn13）的细胞，该蛋白可与UCHL5结合但对蛋白酶体相互作用有缺陷。trRpn13细胞显示蛋白酶体结合的泛素化蛋白水平降低，表明蛋白酶体上hRpn13功能的丧失不能被其他两个专用底物受体（hRpn1和hRpn10）完全补偿。先前的研究表明，全长hRpn13的缺失会导致UCHL5的相应降低。我们发现在trRpn13细胞中UCHL5水平未改变，但hRpn11在ΔhRpn13和trRpn13中升高细胞，也许是由于细胞压力。尽管人细胞中有约90个DUB，除了在蛋白酶体上还有UCHL5外，还有两个，但我们发现从HCT116细胞中缺失UCHL5会导致全细胞提取物和蛋白酶体中泛素化蛋白水平的增加，这表明UCHL5的活性不能由其他DUB完全承担。我们还报告了抗癌分子RA190，它与hRpn13和UCHL5共价结合，需要hRpn13 Pru而非UCHL5来产生细胞毒性。