Regulation of Proteasome Activity by Ubp6 and Hul5

我们之前发现了一组蛋白酶体成分，它们在很大程度上没有被识别出来 这是因为它们是通过传统提纯过程中使用的高盐从蛋白酶体中释放出来的。我们 现在建议更狭隘地关注两个这样的因素：泛素蛋白连接酶Hul5和Ubp6，a 脱泛素酶。我们发现Ubp6还有第二个非常意想不到的功能：非催化 抑制蛋白质降解。此外，当蛋白酶体被UbpG抑制时，另一种 蛋白酶体的去泛素化酶Rpn11被抑制。由于Ubp6修剪泛素链 逐渐地，当Rpn11整体移除链时，Ubp6的存在在模式中施加了切换 底物去泛素化。有趣的是，Ubp6也被发现通过一种新的泛素来调节 压力反应。 我们发现，Hul5可以延长蛋白酶体结合的泛素结合物的链，作用于 特别是在可降解的底物上，如细胞周期蛋白B。因此，Hu！5突变株对许多 底物。UbpG增强了Hul5与蛋白酶体的结合，并且它们之间的功能对立是 提示是通过Hu！5零突变抑制ubp6。此外，Hul5共轭的产物可以是 被蛋白酶体结合的Ubp6快速分解。我们建议，通过调节 蛋白酶体结合的泛素链，Hul5和Ubp6活性的平衡调节底物承诺 降级。Ubp6引起的降解延迟可能有助于确保去泛素化 Ubp6的活性和Hul5的泛素连接活性有足够的时间重塑底物结合 泛素链。 我们将更充分地表征Ubp6对非催化蛋白酶体的抑制作用，这得益于一个定量的 降解实验，并确定其抑制机制。我们最近发现，绿巨人5也有一个 对蛋白酶体功能的显著非催化影响，这将得到进一步的表征。我们还将 验证以下假设：UbpG的去泛素化活性也介导了蛋白酶体抑制； Hul5和Ubp6之间存在功能上的对立，导致泛素链处于 当与蛋白酶体结合时处于动态状态。蛋白酶体是一种关键的细胞调节因子，具有重要意义。 为了人类健康，蛋白酶体抑制剂正在临床上用于治疗越来越多的癌症。 因此，通过对这种酶的更深入的了解，我们的研究可能会促进 其他有用的药物。

We previously identified an ensemble of proteasome components which had been largely unrecognized for years because they are released from proteasomes by the high salt used in conventional purifications. We now propose to focus more narrowlyon two such factors: Hul5, a ubiquitin-protein ligase, and Ubp6, a deubiquitinating enzyme. We have found that Ubp6 has a second, highly unexpected function: noncatalytic inhibition of protein degradation. Moreover,when the proteasome is inhibited by UbpG, the other deubiquitinating enzyme of the proteasome, Rpn11, is suppressed. Since Ubp6 trims ubiquitin chains progressively, while Rpn11 removes chains en bloc, the presence of Ubp6 imposes a switch in the modeof substrate deubiquitination. Interestingly, Ubp6 was also found to be regulated through a novel ubiquitin stress response. We found that Hul5 serves to extend the chains of proteasome-bound ubiquitin conjugates, acting in particular on degradative substrates such as cyclin B. Accordingly, hu!5 mutants show stabilization of many substrates. Hul5 binding to proteasomes is enhanced by UbpG, and functional opposition between them is suggested by suppression of ubp6 by the hu!5 null mutation. Moreover,products of Hul5 conjugation can be rapidly disassembled by proteasome-bound Ubp6. We propose that, by regulating the lengths of proteasome-bound ubiquitin chains, the balance of Hul5 and Ubp6 activity regulates substrate commitment to degradation. The degradation delay caused by Ubp6 might serve to ensure that the deubiquitinating activity of Ubp6 and the ubiquitin-ligating activity of Hul5 have adequate time to refashion substrate-bound ubiquitin chains. We will more fully characterize noncatalytic proteasome inhibition by Ubp6 with the benefit of a quantitative degradation assay, and determine the mechanism of inhibition. We recentlyfound that Hul5 also has a prominent noncatalytic influence on proteasomefunction, and this will be further characterized. We will also test the following hypotheses: that UbpG's activity of deubiquitination also mediates proteasome inhibition; and that there is a functional opposition between Hul5 and Ubp6, resulting in ubiquitin chains being in a dynamic state when bound to the proteasome. The proteasome is a key cellular regulator and is important for human health, as proteasome inhibitors are in clinical use in the treatment of a growing list of cancers. Thus, by providing a deeper understanding of this enzyme, our studies may facilitate the development of other useful drugs.