Regulation of Proteasome Activity by Ubp6 and Hul5

描述（由申请人提供）：我们之前鉴定了一组蛋白酶体成分，这些成分多年来基本上未被识别，因为它们在常规纯化中使用高盐从蛋白酶体中释放出来。我们现在建议更狭隘地关注两个这样的因素：Hul5，一种泛素蛋白连接酶，Ubp6，一种去泛素化酶。我们发现Ubp6具有第二个非常意想不到的功能：非催化性抑制蛋白质降解。此外，当蛋白酶体被UbpG抑制时，蛋白酶体的另一种去泛素化酶Rpn11也被抑制。由于Ubp6逐渐修剪泛素链，而Rpn11整体去除链，Ubp6的存在在底物去泛素化模式中施加了一个开关。有趣的是，Ubp6也被发现通过一种新的泛素应激反应来调节。我们发现Hul5可以延长蛋白酶体结合的泛素缀合物链，特别是作用于降解底物，如细胞周期蛋白b。5个突变体显示出许多底物的稳定性。UbpG增强了Hul5与蛋白酶体的结合，并通过Hul5零突变抑制ubp6提示它们之间的功能对立。此外，Hul5偶联产物可以被蛋白酶体结合的Ubp6快速分解。我们提出，通过调节蛋白酶体结合的泛素链的长度，Hul5和Ubp6活性的平衡调节底物的降解承诺。Ubp6导致的降解延迟可能是为了确保Ubp6的去泛素化活性和Hul5的泛素连接活性有足够的时间来重塑底物结合的泛素链。我们将通过定量降解实验更全面地表征Ubp6对非催化性蛋白酶体的抑制作用，并确定其抑制机制。我们最近发现Hul5对蛋白酶体功能也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；Hul5和Ubp6之间存在功能上的对立，导致泛素链在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剂，对人类健康很重要，因为蛋白酶体抑制剂在临床中用于治疗越来越多的癌症。因此，通过对这种酶提供更深入的了解，我们的研究可能促进其他有用药物的开发。

DESCRIPTION (provided by applicant): 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 narrowly on two such factors: Hul5, an 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 mode of 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 hul5 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 recently found that Hul5 also has a prominent noncatalytic influence on proteasome function, 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.