AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Tufts University, Boston, MA, United States of America.
Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Waltham, MA, United States of America.
PLoS One. 2019 Nov 8;14(11):e0225145. doi: 10.1371/journal.pone.0225145. eCollection 2019.
USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated β-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect β-catenin, demonstrating USP14 but not UCHL5 has a specific effect on β-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides β-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.
USP14 是一种与蛋白酶体相关的半胱氨酸蛋白酶去泛素化酶，在蛋白酶体降解中发挥重要的催化和别构作用。USP14 的抑制作用被认为是一种治疗策略，可以加速神经退行性疾病中聚集倾向蛋白的降解，并抑制蛋白酶体功能以诱导癌症中的细胞凋亡。在这里，我们使用小分子抑制剂和无活性的 USP14 C114A 突变体在哺乳动物细胞中研究了 USP14 抑制的影响。这些抑制剂和 USP14 C114A 都没有对 HEK293T 细胞中的 TDP-43、tau 或 α-突触核蛋白水平产生一致或显著的影响。然而，USP14 C114A 导致泛素化蛋白的大量积累，这些蛋白通过泛素免疫沉淀分离，并通过质谱鉴定。在这些蛋白中，我们通过免疫印迹和免疫沉淀实验证实，表达 USP14 C114A 的细胞中β-连环蛋白的泛素化积累。USP14 C114A 的蛋白酶体结合结构域是其对泛素化蛋白影响所必需的。UCHL5 是另一种与蛋白酶体相关的半胱氨酸蛋白酶去泛素化酶。有趣的是，UCHL5 C88A 的无活性突变体也导致 HEK293T 细胞中泛素化蛋白的积累，但不影响β-连环蛋白，表明 USP14 而不是 UCHL5 对β-连环蛋白具有特异性影响。我们使用泛素免疫沉淀和质谱鉴定在表达 UCHL5 C88A 的细胞中积累的泛素化蛋白，这些蛋白主要与在表达 USP14 C114A 的细胞中鉴定的蛋白不同。在鉴定的蛋白中，有许多是公认的蛋白酶体底物和蛋白酶体亚基。除了β-连环蛋白，我们还通过免疫印迹验证了 UCHL5 C88A 抑制其自身去泛素化，USP14 C114A 抑制两个蛋白酶体亚基 PSMC1 和 PSMD4 的去泛素化。总之，我们的数据表明，USP14 和 UCHL5 可以在蛋白酶体上去泛素化不同的底物，并调节与蛋白酶体功能紧密相关的自身泛素化。
