Studies of Usp14 and the Ubiquitin Stress Response

描述（由申请人提供）：蛋白酶体是泛素依赖性降解途径中的关键酶。它对人体健康很重要，并负责各种基本细胞功能的适当调节。从历史上看，很少有人关注蛋白酶体是如何调节的。我们最近对酵母的研究发现了蛋白酶体调节的意想不到的机制。这两种机制涉及一种与蛋白酶体相关的去泛素化酶Ubp6，其哺乳动物同源物是Usp14:(1) Ubp6抑制蛋白酶体的活性；(2)Ubp6的表达，以及蛋白酶体上Ubp6的量，受泛素水平的负调控。这两种机制可能是相关的，因为低泛素水平诱导Ubp6，然后抑制蛋白酶体活性，从而减少稀缺泛素的破坏。我们的初步数据表明，正如我们在酵母中的发现所预测的那样，Usp14抑制哺乳动物蛋白酶体。在Aim 1中，我们将利用纯化的蛋白酶体和纯化的Usp14以及现有的Usp14无效MEF细胞系，表征哺乳动物中这种抑制的机制及其对细胞功能的影响。在Aim 2中，我们将尝试分离泛素应激反应的分子机制。我们提出的初步数据表明，我们首先在酵母中发现的泛素应激反应也可能在哺乳动物中起作用。我们将使用酵母和哺乳动物系统来剖析这种反应。大量的疾病与蛋白质错误折叠及其病理作用有关。众所周知，错误折叠的蛋白质是泛素-蛋白酶体途径的首选底物。因此，在许多疾病状态下，错误折叠的蛋白质可能会对泛素-蛋白酶体系统施加异常高的负荷。在这种情况下，可能有必要通过补偿性调节机制来维持系统的功能。因此，本文将首次在哺乳动物中探索的调节机制类型可能与多种疾病的发病机制有关。蛋白酶体是控制数百种蛋白质水平的关键酶，因此对多种基本细胞功能的适当调节负责。我们最近对酵母的研究发现了这种酶本身被控制的意想不到的机制。这笔拨款主要是为了测试这些有趣的新机制是否适用于哺乳动物。如果是这样，这项工作可能会推进我们对人类健康的理解，因为泛素-蛋白酶体系统在癌症和神经退行性疾病中都起着重要作用。

DESCRIPTION (provided by applicant): The proteasome is a key enzyme in the ubiquitin-dependent pathway of degradation. It is important in human health and is responsible for proper regulation of a wide variety of basic cell functions. Historically, little attention has been given to how the proteasome is regulated. Our recent studies in yeast have identified unanticipated mechanisms of proteasome regulation. The two mechanisms involve a proteasome-associated deubiquitinating enzyme, Ubp6, whose mammalian ortholog is Usp14: (i) Ubp6 inhibits the activity of the proteasome, and (ii) the expression of Ubp6, and in turn the amount of Ubp6 on the proteasome, are negatively regulated by ubiquitin levels. These two mechanisms are likely related, in that low ubiquitin levels induce Ubp6, which then inhibits proteasome activity, which has the effect of reducing the destruction of scarce ubiquitin. Our preliminary data indicate that, as predicted from our findings in yeast, Usp14 inhibits mammalian proteasomes. In Aim 1, we will characterize the mechanism of this inhibition in mammals and its effects on cell function, using purified proteasomes and purified Usp14 as well as an existing line of usp14 null MEF cells. In Aim 2, we will attempt to isolate the molecular machinery of the ubiquitin stress response. We present preliminary data that the ubiquitin stress response, which we first identified in yeast, may operate in mammals as well. We will use both yeast and mammalian systems to dissect the response. A large number of diseases involve protein misfolding and its pathological effects. It is well appreciated that misfolded proteins are preferred substrates for the ubiquitin-proteasome pathway. Thus, in many disease states, misfolded proteins may impose an unusually high load on the ubiquitin-proteasome system. In such instances, it may be necessary for the system to maintain function through compensatory regulatory mechanisms. Therefore, the types of regulatory mechanisms that will be probed here for the first time in mammals may potentially be relevant to the pathogenesis of multiple diseases. PUBLIC HEALTH RELEVANCE The proteasome is a key enzyme that controls the levels of hundreds of proteins and thus is responsible for the proper regulation of a wide variety of basic cell functions. Our recent studies in yeast have identified unanticipated mechanisms by which this enzyme is itself controlled. The grant is designed mainly to test whether these interesting new mechanisms may apply to mammals. If so, the work may advance our understanding of human health, because the ubiquitin-proteasome system plays important roles in both cancer and neurodegenerative disease.