Mechanisms regulating proteasomal substrate degradation

蛋白酶体底物降解的调节机制

基本信息

批准号：
8875711
负责人：
David Matthew Smith
金额：
$ 28.31万
依托单位：
WEST VIRGINIA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8875711
关键词：
26S proteasome ATP Hydrolysis ATP phosphohydrolase Active Sites Affect Aging Allosteric Regulation Binding Biochemical Biological Biological Models Cell model Cell physiology Cells Complex Data Degenerative Disorder Degradation Pathway Development Disease Drug Design Enzymes Event Family Foundations Goals Health In Vitro Individual Knowledge Life Link Malignant Neoplasms Mechanics Mission Modeling Molecular Molecular Machines Molecular Models Neurodegenerative Disorders Outcome Pathway interactions Peptides Positioning Attribute Process Protein Binding Proteins Public Health Recruitment Activity Regulation Research Role Structure System Therapeutic Agents Time Ubiquitin Ubiquitinated Protein Degradation Work Yeast Model System base biophysical techniques genetic regulatory protein human disease innovation insight molecular modeling multicatalytic endopeptidase complex novel novel therapeutics p97 ATPase particle protein degradation protein function reconstitution therapeutic development tool yeast genetics

项目摘要

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.

描述（由申请人提供）：泛素蛋白酶体系统基本上调节了每个细胞过程及其不调节可能引起或增强疾病。 20S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。但是，我们的知识存在差距，即如何识别泛素化蛋白，然后注入20年代以进行降解。已经证明了两种不同的ATP依赖性复合物可以催化泛素化蛋白的降解，19S调节粒子和P97（VCP/CDC48）。 19S与20年代合作制作26S蛋白酶体；但是，我们不了解19S的ATPases-uses ATP的分子机械环如何结合和转移底物为20s。关于p97如何催化蛋白质降解，包括它是否与蛋白酶体相关，甚至了解较少的理解。我们的长期目标是了解如何调节蛋白质降解并开发专门针对这些调节机制的调节剂，这些机制可以用作研究工具或治疗剂。该应用的总体目标是实现这一长期目标的下一步，是阐明蛋白酶体ATPases和P97在分子水平上的功能如何促进蛋白质降解。该目标的理由是，需要详细的分子模型，即需要如何处理泛素化蛋白以降解，以了解它们如何涉及疾病。申请的目标将通过追求两个具体目标来实现。第一个 AIM将确定蛋白酶体ATPases中的变构调节如何控制ATP水解的位置和时机以正确协调底物降解。利用酵母遗传学支持的古细胞和真核模型系统，将采用各种生化和生物物理方法。第二个目标将决定p97如何催化蛋白酶体降解底物降解。将使用体外重构系统采用类似的酶学方法，并且将在哺乳动物和酵母模型系统中评估推定的P97-20相互作用的重要性。这种方法具有创新性，因为我们已经生成了一种新颖的实验系统，该系统将使我们能够研究这些酶的特定作用和功能，并以新颖的方式将这些发现应用于细胞模型，以确定其功能和生物学作用。预计这些结果将产生重要的积极影响，因为它们确定了UPS的调节特征，这些特征是我们不存在泛素依赖性蛋白质降解所缺少的。这一贡献很重要，因为对这些分子机器如何催化蛋白质降解对于理解如何在癌症，神经退行性疾病和衰老等疾病中误导这一关键过程至关重要。这种见解将为发展新的治疗策略的发展奠定基础，以特别抑制或激活这些单独的退化途径。