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: 8104087
• 负责人: Kylie J. Walters
• 金额: $ 32.67万
• 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/8104087
• 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）：必须严格调节蛋白质的蛋白质降解，因为它控制了从细胞周期进程到细胞死亡的事件。 26S蛋白酶体由20S催化核心颗粒（CP）组成，该核心粒子（CP）由19S调节粒子（RP）封顶。尽管CP包含负责蛋白质水解蛋白底物的酶活性，但对蛋白质降解的承诺由RP成分确定，RP成分在通过其通过之前识别和处理底物进入CP。对于大多数蛋白酶体的底物，泛素化是其降解的先决条件，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。随后，底物通过三种去泛素化酶去泛素化，并由ATPases的异己甲环子展开。这些活动使底物通过狭窄的腔室进入蛋白酶体CP的催化中心。由于并非所有将停滞在蛋白酶体中的泛素化蛋白都被降解，因此在理解RP中底物处理的机械细节方面非常感兴趣。我们的长期目标是定义蛋白酶体的19S调节粒子如何处理底物。我们通过对RPN13和S5A的研究将该提案重点放在此过程的第一阶段，即底物识别和去泛素化。我们研究如何将RPN13停靠到蛋白酶体中，并追求初步数据，以表明它被此本地化激活。我们已经设计了一种新的策略，可以在RP的背景下研究RPN13和S5A，并测试其与泛素化底物的协调结合的工作模型。为了实现我们的研究目标，我们使用功能分析，包括测试纯化蛋白酶体物种和泛素链链链链蛋白链的泛素结合能力的测定方法，以及各种生物物理方法，尤其是NMR光谱法。最终，拟议的研究的完成将提供有关RPN13和S5A所假定的不同作用的基本信息，这些信息不仅仅是将泛素化的蛋白质停靠到蛋白酶体中。 公共卫生相关性：该提案着重于蛋白酶体，蛋白酶体是一种在适当时间进行适当细胞功能的生物机械。该蛋白酶体是针对神经退行性疾病和癌症的主要治疗靶标，其对Velcade的一般抑制作用用于治疗多发性骨髓瘤。该提案的结果将提供有关蛋白酶体如何降解其底物的基本信息以及确定泛素化蛋白是否为降解的涉及的因素。