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，其失调可导致或加剧疾病。20S核心蛋白酶体是这一过程的终点，其内部活性位点将蛋白质降解为小肽。然而，对于如何识别泛素化蛋白并将其注入20S中进行降解，我们的知识还存在空白。两种不同的atp依赖复合物已被证明可以催化泛素化蛋白的降解，即19S调节颗粒和P97 （VCP/CDC48）。19S与20S结合形成26S蛋白酶体；然而，我们并不了解19S的分子机制——它的ATP环——是如何利用ATP结合底物并将其转运到20S中的。关于P97如何催化蛋白质降解，包括它是否与蛋白酶体相关，人们了解的就更少了。我们的长期目标是了解蛋白质降解是如何被调节的，并开发专门针对这些调节机制的调节剂，这些调节剂可以用作研究工具或治疗剂。该应用程序的总体目标是阐明蛋白酶体atp酶和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，需要详细的泛素化蛋白如何加工降解的分子模型，以了解它们的错误调节如何参与疾病。应用程序的目标将通过追求两个具体目标来实现。第一个

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.