Abstract：
The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.
Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive.
We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37.
We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor.
This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.
Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive.
We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37.
We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor.
This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.
摘要：
蛋白酶体上（多）泛素链的去除是蛋白质降解途径中的关键步骤，该途径决定了哪些蛋白质被降解并最终决定了细胞命运。三种不同的去泛素化酶（DUB）与人类蛋白酶体相关，PSMD14(RPN11),USP14和UCH37(UCHL5)。然而,这些蛋白酶体DUB的功能作用和特异性仍然难以捉摸。为了揭示蛋白酶体相关DUB的特异性，我们使用基于SILAC的定量泛素组学研究基于CRISPR-Cas9的敲除这些DUB对动态细胞泛素组的影响。我们观察到在去除USP14或UCH37时对全局泛素组的不同影响，而同时去除两个DUB表明功能冗余比先前预期的要少。我们还通过比较野生型与USP14/UCH37双敲除细胞的治疗，研究了小分子抑制剂b-AP15是否具有特异性靶向USP14和UCH37的潜力。引人注目的是，观察到广泛和严重的脱靶效应，质疑这种抑制剂的所谓特异性。总之,这项工作提供了对蛋白酶体相关DUB功能的新见解，并说明了深入的泛素组学筛选DUB和DUB调节化合物活性的能力。
简介：蛋白酶体上（多）泛素链的去除是蛋白质降解途径中的关键步骤，该途径决定了哪些蛋白质被降解并最终决定了细胞命运。三种不同的去泛素化酶（DUB）与人类蛋白酶体相关，PSMD14/RPN11、USP14和UCH37/UCHL5。然而,这些蛋白酶体DUB的功能作用和特异性仍然难以捉摸。
我们已经应用基于SILAC的定量泛素组学来研究基于CRISPR-Cas9的敲除这些DUB中的每一个对动态细胞泛素组的影响。此外，我们研究了小分子抑制剂b-AP15的功能,它有可能特异性靶向USP14和UCH37.
我们报告了在去除USP14或UCH37时对泛素组和蛋白酶体清除蛋白质的能力的不同影响，而同时去除两个DUB表明冗余比以前预期的要少。此外,对于b-AP15,观察到广泛且严重的脱靶效应,这对该抑制剂的所谓特异性提出质疑.
这项工作提出了对蛋白酶体相关DUB功能的新见解，并说明了深入的泛素组学筛选DUB和DUB调节化合物活性的能力。
简介：蛋白酶体上（多）泛素链的去除是蛋白质降解途径中的关键步骤，该途径决定了哪些蛋白质被降解并最终决定了细胞命运。三种不同的去泛素化酶（DUB）与人类蛋白酶体相关，PSMD14/RPN11、USP14和UCH37/UCHL5。然而,这些蛋白酶体DUB的功能作用和特异性仍然难以捉摸。
我们已经应用基于SILAC的定量泛素组学来研究基于CRISPR-Cas9的敲除这些DUB中的每一个对动态细胞泛素组的影响。此外，我们研究了小分子抑制剂b-AP15的功能,它有可能特异性靶向USP14和UCH37.
我们报告了在去除USP14或UCH37时对泛素组和蛋白酶体清除蛋白质的能力的不同影响，而同时去除两个DUB表明冗余比以前预期的要少。此外,对于b-AP15,观察到广泛且严重的脱靶效应,这对该抑制剂的所谓特异性提出质疑.
这项工作提出了对蛋白酶体相关DUB功能的新见解，并说明了深入的泛素组学筛选DUB和DUB调节化合物活性的能力。
