Mechanisms regulating proteasomal substrate degradation

描述（由申请人提供）：泛素蛋白酶体系统基本上调节每一个细胞过程，其失调可导致或加重疾病。20 S核心蛋白酶体是该过程的终点，其内部活性位点将蛋白质降解为小肽。然而，我们对泛素化蛋白如何被识别并注入20 S进行降解的知识存在空白。两种不同的ATP依赖性复合物已被证明可以催化泛素化蛋白的降解，19 S调节颗粒和P97（VCP/CDC 48）。19 S与20 S结合形成26 S蛋白酶体;然而，我们不了解19 S的分子机制--它的ATP酶环--如何利用ATP结合底物并将其转运到20 S中。关于P97如何催化蛋白质降解的了解甚至更少，包括它是否与蛋白酶体相关。我们的长期目标是了解蛋白质降解是如何调节的，并开发专门针对这些调节机制的调节剂，可用作研究工具或治疗剂。本申请的总体目标是阐明蛋白酶体ATP酶和P97如何在分子水平上发挥作用以促进蛋白质降解，这是实现该长期目标的下一步。这一目标的基本原理是，需要详细的分子模型来了解泛素化蛋白如何被降解，以了解它们的失调如何参与疾病。申请的目的将通过追求两个具体目标来实现。第一 目的是确定蛋白酶体ATP酶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。各种生物化学和生物物理学的方法将利用酵母遗传学支持的古菌和真核模型系统。第二个目标将确定P97如何催化蛋白酶体的底物降解。将采用类似的酶学方法使用体外重建系统，并将在哺乳动物和酵母模型系统中评估推定的P97- 20 S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的实验系统，使我们能够研究这些酶的特定作用和功能，并以新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作用。这些结果预计将产生重要的积极影响，因为它们确定了UPS的调节特征，这些特征在我们对泛素依赖性蛋白质降解的理解中缺失。这一贡献是重要的，因为了解这些分子机器如何催化蛋白质降解对于了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节至关重要。这些见解将为开发新的治疗策略以特异性抑制或激活这些单独的降解途径奠定基础。

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.