Structures of Proteasome Activators

蛋白酶体激活剂的结构

• 批准号: 7821252
• 负责人: CHRISTOPHER P. HILL
• 金额: $ 33.52万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7821252
• 关键词: 26S proteasome; ATP phosphohydrolase; Architecture; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biochemistry; Biological; C-terminal; Cell Nucleus; Complex; Cryoelectron Microscopy; Crystallization; Cytosol; Data; Development; Disease; Distant; Electron Microscopy; Electrons; Engineering; Enzymes; Eukaryota; Family; Future; Goals; Heterogeneity; Human; Hydrolysis; Individual; Laboratories; Left; Life; Light; Mammals; Mass Spectrum Analysis; Mediating; Modeling; Molecular Conformation; Mutagenesis; Outcome; Pathway interactions; Peptide Hydrolases; Peptides; Positioning Attribute; Preparation; Process; Protein Subunits; Proteins; Protocols documentation; Published Comment; Publishing; Recombinants; Regulation; Reporting; Resolution; Scheme; Structure; Structure-Activity Relationship; Surface; System; Testing; Trypanosoma; Work; Yeasts; base; cancer therapy; design; genetic analysis; in vivo; inhibitor/antagonist; macromolecular assembly; multicatalytic endopeptidase complex; mutant; novel therapeutics; programs; protein degradation; protein folding; public health relevance; reconstitution; reconstruction; scaffold; yeast genetics

DESCRIPTION (provided by applicant): A 700 kDa protease called the 20S proteasome (20S) is essential for eukaryotic life. Isolated 20S are inactive because the gate to their internal catalytic chamber is closed. Substrates/products enter/exit the 20S interior via activator complexes that bind to the 20S ends and open the entrance gate. Three classes of activator have been identified: 11S, Blm10, and 19S. Previously we focused on 11S activators and determined 1.1 MDa, 42 subunit structures of 11S complexes with both yeast and archaeal 20S. Remarkably, the archaeal 20S complex crystals diffracted to better than 2.0 A resolution. Based upon these structures, we proposed mechanisms of binding and gate opening, and confirmed these models through mutagenesis, biochemistry, and structure determination of mutant 20S:11S complexes. We further suggested that 19S employs a similar mechanism of binding and stabilizes the same open conformation, and we validated these proposals with mutagenic and biochemical studies. Building on this progress, our future studies seek a detailed understanding of all three classes of proteasome activator. Aim 1 - Blm10. Preliminary data include development of expression and purification protocols that yield active Blm10-20S complex, clarification of data from yeast genetics, determination of an 18 A structure by electron cryomicroscopy, crystallization, and preliminary crystal structure determination at 3.5 A resolution. The emerging structure reveals an extensive contact surface and disordering of the 20S gate to a partially open conformation. An unexpected bonus is that the structure suggests a detailed model for how 19S activator binds and opens the 20S gate. Aim 2 - 11S. Our well-defined PA26:20S system is being exploited to design chimeric constructs that will provide detailed models of how the highly diverged mammalian 11S activators bind and activate proteasomes. This approach will also advance detailed models of how 19S/PAN activator subunits bind and open the proteasome gate. Preliminary crystals have been prepared. Aim 3 - S19. Preparations of the 900 kDa multisubunit 19S activator and its 2.5 MDa complex with 20S (the 26S proteasome) suffer from heterogeneity and are therefore being approached by recombinant expression and reconstitution of functionally significant subcomplexes. Several subcomplexes have been prepared, one of which has yielded preliminary crystals. PUBLIC HEALTH RELEVANCE: Proteasomes are large macromolecular assemblies that are essential in eukaryotes and are attractive targets for the development of new therapeutics, especially for the treatment of cancers. We aim to advance understanding of proteasome regulation through biochemical, genetic, and structural studies of their biological activators.

描述（由申请人提供）：一种称为20S蛋白酶体（20s）的700 kDa蛋白酶对于真核生活至关重要。孤立的20s是不活跃的，因为它们的内部催化室的门被关闭。底物/产品通过与20s结合并打开入口门的激活剂复合物进入/退出20S内部。已经确定了三类激活剂：11S，BLM10和19S。以前，我们专注于11S激活剂，并确定了1.1 MDA，42个具有酵母菌和20s的11S络合物的亚基结构。值得注意的是，古细菌20S复合物晶体衍射到优于2.0分辨率。基于这些结构，我们提出了结合和栅极开放的机制，并通过诱变，生物化学和突变体20S：11S复合物的结构测定确认了这些模型。我们进一步建议，19S采用了类似的结合机制，可以稳定相同的开放构象，我们通过诱变和生化研究验证了这些建议。在这一进步的基础上，我们的未来研究寻求对所有三类蛋白酶体激活剂的详细理解。 AIM 1 -BLM10。初步数据包括产生活跃的BLM10-20复合物的表达和纯化方案的开发，从酵母遗传学中阐明数据，通过电子冷冻机镜检查，结晶和初步的晶体结构在3.5 A分辨率下确定18 A结构。新兴结构揭示了20S门的广泛接触表面和部分开放构象的无序。意外的奖励是该结构为19S激活剂如何绑定并打开20S门的详细模型。目标2-11S。我们定义明确的PA26：20S系统被利用为设计嵌合构建体，这些嵌合构建体将提供高度差异的哺乳动物11S激活剂如何结合和激活蛋白酶体的详细模型。这种方法还将推进19s/pan激活子亚基如何结合和打开蛋白酶体门的详细模型。初步晶体已经制备。目标3 -S19。 900 kDa多亚基19S激活剂及其2.5 MDA复合物具有20S（26S蛋白酶体）的制剂，因此具有异质性，因此可以通过重组表达和重组表达和重组的功能性显着亚复合物来接近。已经制备了几个子复合物，其中一种产生了初步晶体。公共卫生相关性：蛋白酶体是在真核生物中必不可少的大型大分子组件，是开发新疗法的有吸引力的靶标，尤其是用于治疗癌症。我们旨在通过其生物激活剂的生化，遗传和结构研究来提高对蛋白酶体调节的了解。