Mechanisms regulating proteasomal substrate degradation

描述(申请人提供)：泛素蛋白酶体系统基本上调节每一个细胞过程，它的错误调节可能导致或加重疾病。20S核心蛋白酶体是这一过程的终点，其内部活性部位将蛋白质降解成小肽。然而，我们对泛素化蛋白质是如何被识别，然后注入到20s中进行降解的了解还存在一个空白。两种不同的依赖于ATP的复合体催化泛素化蛋白质的降解，19S调节颗粒和P97(VCP/CDC48)。19年代与20年代联系在一起，形成了26S蛋白酶体；然而，我们不知道19年代的分子机制--它的ATPase环--如何利用ATP结合和转移底物到20年代。关于P97如何催化蛋白质降解，包括它是否与蛋白酶体相关，人们更是知之甚少。我们的长期目标是了解蛋白质降解是如何受到调控的，并开发专门针对这些调控机制的调节剂，这些调节剂可以用作研究工具或治疗剂。这项应用的总体目标是阐明蛋白酶体ATPase和P97如何在分子水平上促进蛋白质降解，这是实现这一长期目标的下一步。这一目标的基本原理是，需要建立泛素化蛋白质如何被处理以进行降解的详细分子模型，以了解它们的错误调控如何与疾病有关。申请的目标将通过追求两个具体目标来实现。第一 AIM将确定蛋白酶体ATPase中的变构调节如何控制ATP水解的位置和时间，以适当地协调底物降解。将利用酵母遗传学支持的古生物和真核模型系统，采取各种生化和生物物理方法。第二个目标将确定P97如何通过蛋白酶体催化底物降解。使用体外重组系统将采取类似的酶学方法，并将在哺乳动物和酵母模型系统中评估假定的P97-20S相互作用的重要性。这种方法是创新的，因为我们已经产生了一种新颖的实验系统，它将使我们能够研究这些酶的特定角色和功能，并以新颖的方式将这些发现应用于细胞模型，以确定它们的功能和生物学角色。这些结果预计将产生重要的积极影响，因为它们确定了UPS的监管功能，这些功能是我们对泛素依赖的蛋白质降解的理解所缺失的。这一贡献意义重大，因为了解这些分子机器如何催化蛋白质降解对于理解这一关键过程如何在癌症、神经退行性疾病和衰老等疾病中受到错误调控至关重要。这些见解将为开发新的治疗策略奠定基础，以具体抑制或激活这些单独的降解途径。

DESCRIPTION (provided by applicant): The Ubiquitin Proteasome System regulates essentially every cellular process and its misregulation can cause or potentiate disease. The 20S core proteasome is the endpoint of this process and its internal active sites degrade proteins to small peptides. However, there is a gap in our knowledge about how ubiquitinated proteins are recognized and then injected into the 20S for degradation. Two different ATP-dependent complexes have been shown to catalyze the degradation of ubiquitinated proteins, the 19S regulatory particle and P97 (VCP/CDC48). The 19S associates with the 20S to make the 26S proteasome; however, we do not understand how the 19S's molecular machinery-its ring of ATPases-uses ATP to bind and translocate substrates into the 20S. Even less is understood about how P97 catalyzes protein degradation including whether or not it even associates with the proteasome. Our long-term goal is to understand how protein degradation is regulated and to develop modulators that specifically target these regulatory mechanisms, which can be used as research tools or therapeutic agents. The overall objective of this application, which is the next step toward attaining this long- term goal, is to elucidate how the proteasomal ATPases and P97 function at a molecular level to facilitate protein degradation. The rationale for this objective is that detailed molecular models of how ubiquitinated proteins are processed for degradation are needed to understand how their misregulation is involved in disease. The objective of the application will be attained by pursuing two specific aims. The first aim will determine how allosteric regulation in the proteasomal ATPases control the position and timing of ATP hydrolysis to properly coordinate substrate degradation. Various biochemical and biophysical approaches will be taken utilizing both archaeal and eukaryotic model systems supported by yeast genetics. The second aim will determine how P97 catalyzes substrate degradation by the proteasome. Similar enzymological approaches will be taken using in vitro reconstituted systems, and the importance of a putative P97-20S interaction will be evaluated in mammalian and yeast model systems. This approach is innovative because we have generated a novel experimental system that will allow us to investigate the specific roles and functions of these enzymes and apply these findings to cell models in novel ways to determine their functional and biological roles. These outcomes are expected to have an important positive impact because they identify regulatory features of the UPS that have been missing from our under- standing of ubiquitin-dependent protein degradation. This contribution is significant because an understanding of how these molecular machines catalyze protein degradation is essential for understanding how this critical process can be misregulated in diseases such as cancer, neurodegenerative disease and aging. Such insights will lay the foundation for the development of new therapeutic strategies to specifically inhibit or activate these separate degradation pathways.