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: 8403784
• 负责人: Kylie J. Walters
• 金额: $ 30.53万
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403784
• 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; 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.

描述(申请人提供)：蛋白酶体的蛋白质降解必须受到严格的调控，因为它控制着从细胞周期进展到细胞死亡的一系列事件。26S蛋白酶体由一个20S催化核心颗粒(CP)组成，其两端各有一个19S调节颗粒(RP)。CP包含负责蛋白质底物降解的酶活性，而对蛋白质降解的承诺由RP组分决定，这些组分在底物进入CP之前识别和处理底物。对于大多数蛋白酶体底物，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。底物随后被三种去泛素化酶去泛素化，并通过ATPase的异六聚环展开。这些活动使底物可以通过通往蛋白酶体CP催化中心的狭小腔室进入。由于并不是所有停靠在蛋白酶体中的泛素化蛋白质最终都会被降解，因此人们对了解RP中底物处理的机制细节非常感兴趣。我们的长期目标是定义底物是如何被蛋白酶体的19年代调节颗粒处理的。通过对Rpn13和S5a的研究，我们将这一建议集中在这一过程的第一阶段，即底物识别和去泛素化。我们研究了Rpn13是如何对接到蛋白酶体中的，并寻求表明它被这种定位激活的初步数据。我们设计了一种新的策略，在RP的背景下研究Rpn13和S5a，并测试它们与泛素化底物配位结合的工作模型。为了实现我们的研究目标，我们使用了功能分析，包括测试纯化的蛋白酶体物种的泛素结合能力和Uch37对泛素链的去结合能力，以及各种生物物理方法，特别是核磁共振光谱。最终，这项拟议研究的完成将提供有关Rpn13和S5a所扮演的不同角色的基本信息，这些角色不仅仅是将泛素化的蛋白质对接到蛋白酶体中。

项目成果

Myosin VI Contains a Compact Structural Motif that Binds to Ubiquitin Chains.

• DOI: 10.1016/j.celrep.2016.01.079
• 发表时间: 2016-03-22
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: He F;Wollscheid HP;Nowicka U;Biancospino M;Valentini E;Ehlinger A;Acconcia F;Magistrati E;Polo S;Walters KJ
• 通讯作者: Walters KJ