Characterization of deubiquitylating enzymes in yeast

蛋白质泛素化是一种重要的翻译后修饰，负责靶向蛋白质降解，细胞信号激活，DNA损伤反应和蛋白质运输。去泛素化酶（Deubiquitylating酶，DUBs）通过去除泛素化蛋白中的泛素来消除泛素信号。尽管dub具有重要意义，但人们对其生物学意义知之甚少。为了测试DUBs的酶学特性，我们利用小麦胚芽表达系统对酵母20 DUBs进行了表达和纯化。虽然大多数DUBs不具有链型特异性，但我们发现四个DUBs可以切割泛素前体Ubi4。在蛋白酶体dub中，与以往的研究一致，Rpn11对泛素化底物的降解至关重要，而Ubp6对底物的体外降解具有抑制作用。然而，这两种dub都可以切割K48-和k63 -连接的泛素链，这表明其他因素可能指定蛋白酶体降解信号。我们还发现Ubp6通过其去泛素化活性清除组装前体中的泛素化底物，从而促进蛋白酶体组装。此外，我们应用平行反应监测（PRM）方法定量泛素链并分析DUB突变体。

Protein ubiquitylation is an essential post-translational modification responsible for targeted protein degradation, cellular signaling activation, DNA damage response, and protein trafficking. Deubiquitylating enzymes (DUBs) functions as an eraser of the ubiquitin signals by removing ubiquitin(s) from the ubiquitylated proteins. Despite of their importance, biological significance of DUBs poorly understood. To test enzymatic properties of DUBs, we expressed and purified yeast 20 DUBs by a wheat germ expression system. Although most DUBs did not have chain-type specificities, we found that four DUBs can cleave a ubiquitin precursor Ubi4. Among the proteasomal DUBs, consistently with previous studies, Rpn11 is essential for degradation of the ubiquitinated substrate whereas Ubp6 has an inhibitory effect for the substrate degradation in vitro. However, both DUBs can cleave both K48- and K63-linked ubiquitin chains, suggesting that other factors might specify the proteasomal degradation signal. We also found that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity. Furthermore, we applied parallel reaction monitoring (PRM) method to quantify the ubiquitin chains and analyzed DUB mutants.