NMR Structural Studies of Ubiquitin Receptor Protein Complexes

泛素受体蛋白复合物的 NMR 结构研究

• 批准号: 7990131
• 负责人: Kylie J. Walters
• 金额: $ 18.01万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-02 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990131
• 关键词: 26S proteasome; ATP phosphohydrolase; Affect; Affinity; Amino Acids; Animal Model; Area; Behavior; Binding; Biological Assay; Catalytic Domain; Cell Cycle Progression; Cell Death; Cell physiology; Commit; Complex; Crystallization; Crystallography; DNA Repair; Data; Deubiquitinating Enzyme; Deubiquitination; Docking; Event; Genetic Transcription; Goals; Human Herpesvirus 8; Human Resources; Immunocompetent; Laboratories; Length; Link; Longevity; Lysine; Malignant Neoplasms; Methods; Modeling; Multiple Myeloma; NMR Spectroscopy; Neurodegenerative Disorders; Nucleosome Core Particle; Outcome; Pathway interactions; Peptides; Postdoctoral Fellow; Process; Proteasome Binding; Proteins; Proteolysis; Public Health; Publishing; Reagent; Research; Role; Scaffolding Protein; Site; Staging; Structure; System; Testing; Time; UBD protein; Ubiquitin; Ubiquitination; Velcade; Work; Yeasts; experience; genetic regulatory protein; interest; latency-associated nuclear antigen; multicatalytic endopeptidase complex; novel strategies; particle; protein complex; protein degradation; protein misfolding; public health relevance; receptor; therapeutic target; trafficking; yeast genetics

DESCRIPTION (provided by applicant): Protein degradation by the proteasome must be tightly regulated, as it controls events ranging from cell cycle progression to cell death. The 26S proteasome is composed of a 20S catalytic core particle (CP) that is capped at either end by a 19S regulatory particle (RP). Whereas the CP contains the enzymatic activity responsible for proteolyzing protein substrates, the commitment to protein degradation is determined by RP components, which recognize and process substrates prior to their passage into the CP. For most proteasome substrates, ubiquitination is prerequisite to their degradation, as their initial interaction with the proteasome is through RP ubiquitin receptors, S5a and Rpn13. Substrates are subsequently deubiquitinated by three deubiquitinating enzymes and unfolded by a heterohexomeric ring of ATPases. These activities prepare substrates for entry through a narrow chamber leading to the catalytic center of the proteasome's CP. Since not all ubiquitinated proteins that dock into the proteasome are ultimately degraded, there is a great deal of interest in understanding the mechanistic details of substrate processing in the RP. Our long-term goal is to define how substrates are processed by the 19S regulatory particle of the proteasome. We focus this proposal on the first stage of this process, namely substrate recognition and deubiquitination, through our studies of Rpn13 and S5a. We study how Rpn13 is docked into the proteasome and pursue preliminary data that indicates it to be activated by this localization. We have devised a novel strategy to study Rpn13 and S5a in the context of the RP and test a working model of their coordinated binding to ubiquitinated substrates. To achieve our research goals, we use functional assays, including those that test the ubiquitin binding capacity of purified proteasome species and ubiquitin chain deconjugation by Uch37, as well as a variety of biophysical methods, especially NMR spectroscopy. Ultimately, the completion of the proposed research will provide fundamental information on distinct roles assumed by Rpn13 and S5a that extend beyond simply docking ubiquitinated proteins into the proteasome. PUBLIC HEALTH RELEVANCE: This proposal focuses on the proteasome, a biomachine that removes proteins at the appropriate time for proper cellular function. The proteasome is a major therapeutic target against neurodegenerative diseases and cancer and its general inhibition by Velcade is used to treat multiple myeloma. The outcome of this proposal will afford fundamental information on how the proteasome degrades its substrates and factors involved in determining whether ubiquitinated proteins are fated for degradation.

描述（由申请人提供）：蛋白酶体的蛋白质降解必须受到严格调控，因为它控制着从细胞周期进展到细胞死亡的事件。26S蛋白酶体由一个20S催化核心颗粒（CP）组成，其两端由一个19S调节颗粒（RP）覆盖。尽管CP含有负责蛋白质水解的酶活性，但蛋白质降解的承诺是由RP组分决定的，RP组分在底物进入CP之前识别和处理底物。对于大多数蛋白酶体底物来说，泛素化是它们降解的先决条件，因为它们与蛋白酶体的初始相互作用是通过RP泛素受体S5a和Rpn13。底物随后被三种去泛素化酶去泛素化，并被atp酶的异六聚体环展开。这些活动为底物通过狭窄的腔室进入蛋白酶体CP的催化中心做准备。由于并非所有停靠在蛋白酶体上的泛素化蛋白最终都会被降解，因此了解RP中底物加工的机制细节非常有趣。我们的长期目标是确定蛋白酶体的19S调节颗粒如何处理底物。我们通过对Rpn13和S5a的研究，重点研究了这一过程的第一阶段，即底物识别和去泛素化。我们研究Rpn13如何与蛋白酶体对接，并寻求初步数据，表明它是由这种定位激活的。我们设计了一种新的策略来研究RP背景下的Rpn13和S5a，并测试了它们与泛素化底物协调结合的工作模型。为了实现我们的研究目标，我们使用了功能分析，包括测试纯化蛋白酶体物种的泛素结合能力和Uch37的泛素链解偶，以及各种生物物理方法，特别是核磁共振光谱。最终，该研究的完成将提供Rpn13和S5a所承担的不同作用的基本信息，这些作用不仅仅是将泛素化蛋白对接到蛋白酶体中。