Regulation of Proteasome Activity by Ubp6 and Hul5

我们以前鉴定了一组蛋白酶体组分，这些组分在很大程度上未被识别， 因为它们通过常规纯化中使用的高盐从蛋白酶体中释放出来。我们 现在我建议更严格地关注两个这样的因子：Hul 5，一种泛素蛋白连接酶，和Ubp 6，一种 去泛素化酶我们发现Ubp 6还有第二个意想不到的功能：非催化性 抑制蛋白质降解。此外，当蛋白酶体被UbpG抑制时， 蛋白酶体的去泛素化酶Rpn 11被抑制。由于Ubp 6修剪泛素链， 逐渐地，当Rpn 11整体地去除链时，Ubp 6的存在强制了模式的切换， 底物去泛素化。有趣的是，Ubp 6也被发现通过一种新的泛素调节。 应激反应 我们发现Hul 5可以延长蛋白酶体结合的泛素结合物的链， 特别是在降解底物如细胞周期蛋白B上。因此，Hu！5突变体显示许多稳定 印刷受体. UbpG增强了Hul 5与蛋白酶体的结合，并且它们之间的功能对立是 这是由Hu！5个无效突变。此外，Hul 5缀合的产物可以是 被蛋白酶体结合的Ubp 6迅速分解。我们建议，通过调节 蛋白酶体结合的泛素链，Hul 5和Ubp 6活性的平衡调节底物承诺 到退化。由Ubp 6引起的降解延迟可能有助于确保去泛素化蛋白的表达。 Ubp 6的活性和Hul 5的泛素连接活性有足够的时间重新形成底物结合， 泛素链 我们将更全面地描述Ubp 6对非催化蛋白酶体的抑制作用， 降解试验，并确定抑制机制。我们最近发现Hul 5也有一个 对蛋白酶体功能有显著的非催化影响，这将进一步表征。我们还将 验证以下假设：UbpG的去泛素化活性也介导蛋白酶体抑制; Hul 5和Ubp 6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的动态状态。蛋白酶体是一种关键的细胞调节因子， 对于人类健康，因为蛋白酶体抑制剂在临床上用于治疗越来越多的癌症。 因此，通过提供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.