Ubiquitin chain editing by the mammalian proteasome

描述（申请人提供）：自发现以来，蛋白酶体通常被视为选择降解底物的被动参与者。这一概念最近受到了来自本实验室的许多研究的挑战，这些研究表明蛋白酶体快速编辑泛素链，并且这些过程强烈调节蛋白酶体的输出。我们首先在酵母中研究了这些问题，但现在建议将重点放在哺乳动物上，这方面做得很少。酵母和哺乳动物之间的调节系统不同，因此在哺乳动物中仔细研究这些问题尤为重要。例如，酵母只有一种酶来修剪蛋白酶体上的链，Ubp 6，而哺乳动物有两种：Usp 14（Ubp 6的直系同源物）和Uch 37。此外，我们的研究表明，链修剪对降解速率的影响在哺乳动物中更显着。我们已经开发了一种特异性的小分子Usp 14抑制剂。使用这种抑制剂，在体内和细胞中，我们发现了一个意想不到的强大的作用链修剪调节降解。这种化合物是独一无二的，首次提供了一种提高泛素-蛋白酶体途径输出的方法。值得注意的是，它诱导多种蛋白毒性蛋白质以及氧化蛋白质的更快降解，并提供对蛋白毒性应激的抗性。原则上，可能的最终治疗应用包括数十种已知的疾病，其中有毒蛋白质表达或蛋白质降解不足。对Uch 37的筛选也已完成，涵盖了130，000种化合物，并获得了高度选择性的抑制剂。尽管Usp 14和Uch 37都是修剪链，但它们似乎以不同的方式调节蛋白酶体，原因尚不清楚。为了理解这一点，我们将首先进行体外和模型底物研究，测试底物结合的泛素链的拓扑连接特异性是否提供了一个潜在的代码，可以解释底物特异性的Usp 14和Uch 37及其对降解速率的影响。类似的模型底物工作将评估蛋白质对蛋白酶体展开的敏感性是否与链修剪有关。底物的哪些特征决定了对编辑因子的敏感性，将使用内源性底物进一步分析？我们将使用最先进的质谱方法，在广泛的（如果不是全球的）规模上确定细胞中的哪些底物通过蛋白酶体链编辑进行调节，同时使用抑制剂和小鼠中的基因敲除。内源性底物的相关性质，如它们携带的泛素链类型和它们的泛素受体依赖性，将被表征。编辑系统的最后一个组成部分是Hul 5，一种保守的蛋白酶体相关泛素连接酶。我们已经证明Hul 5延伸了蛋白酶体结合的泛素链，因此对于酵母中的链编辑很重要，在酵母中它拮抗Ubp 6。到目前为止，Hul 5基本上还没有在哺乳动物中进行过研究。这些结果将为蛋白酶体功能提供重要的新见解，并可能与新疗法的开发有关。

DESCRIPTION (provided by applicant): Since its discovery, the proteasome has generally been viewed as a passive player in the selection of substrates for degradation. This notion has recently been challenged by studies, many from this laboratory, showing that proteasomes rapidly edit ubiquitin chains and that these processes strongly regulate proteasome output. We first examined these issues in yeast, but now propose to focus on mammals, where little is being done. The regulatory systems differ between yeast and mammals, making it especially important that these questions are studied carefully in mammals. For example, whereas yeast has a single enzyme that trims chains on the proteasome, Ubp6, mammals have two: Usp14 (the Ubp6 ortholog) and Uch37. Also, our studies indicate that the influence of chain trimming on degradation rates is more dramatic in mammals. We have developed a specific, small-molecule inhibitor of Usp14. Using this inhibitor, both in vivo and in cells, we discovered an unexpectedly powerful role of chain trimming in regulating degradation. This compound is unique in providing, for the first time, a way to enhance output of the ubiquitin-proteasome pathway. Remarkably, it induces faster degradation of multiple proteotoxic proteins as well as oxidized proteins, and provides resistance to proteotoxic stress. Possible eventual therapeutic applications include, in principle, tens of known diseases in which toxic proteins are expressed or protein degradation is deficient. A screen has also been completed for Uch37, covering 130,000 compounds, and a highly selective inhibitor is in hand. Though Usp14 and Uch37 both trim chains, they seem to regulate the proteasome in distinct ways, for unknown reasons. To understand this, we will first carry out in vitro and model substrate studies testing whether the topological linkage specificity of substrate-bound ubiquitin chains provides an underlying code that could explain the substrate specificities of Usp14 and Uch37 and their effects on degradation rates. Similar model substrate work will assess whether a protein's susceptibility to unfolding by the proteasome in linked to chain trimming. What features of the substrate determine susceptibility to editing factors will be analyzed further using endogenous substrates? We will use state of the art mass spectrometry approaches to determine, on a broad, if not global, scale, which substrates in the cell are regulated via proteasomal chain editing, using the inhibitors as well and gene knockouts in mice. Relevant properties of endogenous substrates, such as the type of ubiquitin chain they carry and their ubiquitin receptor dependence, will be characterized. A final component of the editing system is Hul5, a conserved, proteasome-associated ubiquitin ligase. We have shown Hul5 extends proteasome-bound ubiquitin chains and is thus important for chain editing in yeast, where it antagonizes Ubp6. Thus far Hul5 has essentially not been studied in mammals. The results will provide important new insights into proteasome function and might have relevance to the development of novel therapeutics.