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 蛋白酶对于真核生命至关重要。孤立的 20S 不活动，因为其内部催化室的门已关闭。底物/产品通过与 20S 末端结合并打开入口门的激活剂复合物进入/离开 20S 内部。已鉴定出三类激活剂：11S、Blm10 和 19S。之前我们专注于 11S 激活剂，并确定了 11S 与酵母和古菌 20S 复合物的 1.1 MDa、42 个亚基结构。值得注意的是，古菌 20S 复合晶体的衍射分辨率优于 2.0 A。基于这些结构，我们提出了结合和门打开的机制，并通过突变20S:11S复合物的诱变、生物化学和结构测定证实了这些模型。我们进一步建议 19S 采用类似的结合机制并稳定相同的开放构象，并且我们通过诱变和生化研究验证了这些建议。在此进展的基础上，我们未来的研究将寻求对所有三类蛋白酶体激活剂的详细了解。目标 1 - Blm10。初步数据包括产生活性 Blm10-20S 复合物的表达和纯化方案的开发、酵母遗传学数据的澄清、通过电子冷冻显微镜测定 18 A 结构、结晶和 3.5 A 分辨率的初步晶体结构测定。新兴的结构揭示了广泛的接触表面和20S门的无序化，形成部分开放的构象。一个意想不到的好处是，该结构提出了 19S 激活剂如何结合和打开 20S 门的详细模型。目标 2 - 11S。我们明确定义的 PA26:20S 系统被用来设计嵌合结构，该结构将提供高度分化的哺乳动物 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大门的详细模型。已制备出初步晶体。目标 3 - S19。 900 kDa 多亚基 19S 激活剂及其与 20S（26S 蛋白酶体）的 2.5 MDa 复合物的制备存在异质性，因此正在通过重组表达和重建功能上重要的子复合物来实现。已经制备了几种子复合物，其中之一已经产生了初步晶体。公共健康相关性：蛋白酶体是真核生物中必不可少的大型大分子组装体，是开发新疗法（尤其是癌症治疗）的有吸引力的靶标。我们的目标是通过对其生物激活剂的生化、遗传和结构研究来增进对蛋白酶体调节的理解。