Regulation of Alternative 20S Proteasome Assembly under oxidative stress

氧化应激下替代 20S 蛋白酶体组装的调节

• 批准号: 7991841
• 负责人: Mary Joy Kunjappu
• 金额: $ 2.65万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991841
• 关键词: 26S proteasome; Aging; Alzheimer' s Disease; Biogenesis; Biological Assay; Cell Aging; Cell physiology; Cells; Clear Cell; Disease; Ensure; Eukaryotic Cell; Exhibits; Growth; In Vitro; Longevity; Methods; Mitotic; Molecular Chaperones; Neurons; Nucleosome Core Particle; Oxidative Stress; Parkinson Disease; Peptide Hydrolases; Physiological; Positioning Attribute; Proteins; Regulation; Relative (related person); Role; Ubiquitin; Yeasts; age related; in vivo; multicatalytic endopeptidase complex; mutant; oxidative damage; prevent; protein degradation; reconstitution

DESCRIPTION (provided by applicant): Eukaryotic cells have different mechanisms to degrade damaged proteins. The 203 proteasome, which also .forms the core of the larger, ubiquitin-dependent 26S proteasome, appears to have a major role in clearing the cell of oxidatively damaged proteins. Notably, cellular aging is thought to be accelerated by the accumulation of oxidatively damaged proteins. Assembly of the 20S proteasome from its 14 different subunits is directed by proteasome biogenesis-associated factors 3 and 4 (Pba3/Pba4), which help guide the placement of certain subunits into specific positions; without these chaperones, the outer rings of the 20S proteasome are remodeled into an alternative arrangement. Mutant cells lacking Pba3/Pba4 have a growth advantage under conditions known to cause oxidative damage to proteins. In Aim 1, the physiological role of the alternative proteasome will be determined. If the growth advantage of the chaperone mutants is primarily due to enhanced degradation of damaged proteins by alternative 20S proteasomes, then the mutant should exhibit faster degradation rates of these proteins. Increased degradation of such proteins in cells making alternative proteasomes could also potentially increase chronological lifespan. This will be assayed by manipulating the levels of alternative relative to standard proteasomes and growing the resulting strains either with or without oxidative stress over extended periods. Understanding how these chaperones direct the subunit arrangement of the 20S proteasome is difficult to study in vivo due to the lack of early assembly intermediates that can be isolated. Aim 2 proposes an in vitro approach to reconstitute 20S proteasome assembly. This method is amenable to various manipulations that can reveal the Pba3/Pba4 mechanism of action. The composition of the 20S proteasome will be assessed with and without Pba3/Pba4 to determine the underlying method by which these chaperones function. RELEVANCE: Many aging-related diseases, such as Alzheimer's and Parkinson's disease, are characterized by the accumulation of oxidatively damaged proteins. The 20S proteasome appears to have a special role in degrading such aberrant protein species. Therefore, understanding how different forms of the 20S proteasome are assembled, which can have a major impact on how efficiently these damaged proteins are degraded, will be essential for preventing and treating these diseases.

描述（由申请人提供）：真核细胞具有不同的降解受损蛋白质的机制。 203 蛋白酶体也是较大的泛素依赖性 26S 蛋白酶体的核心，似乎在清除细胞中氧化损伤的蛋白质方面发挥着重要作用。值得注意的是，氧化损伤蛋白质的积累被认为会加速细胞衰老。 20S 蛋白酶体由 14 个不同亚基组装而成，由蛋白酶体生物发生相关因子 3 和 4 (Pba3/Pba4) 指导，有助于引导某些亚基放置到特定位置；如果没有这些伴侣，20S 蛋白酶体的外环将被重塑为另一种排列。缺乏 Pba3/Pba4 的突变细胞在已知会导致蛋白质氧化损伤的条件下具有生长优势。在目标 1 中，将确定替代蛋白酶体的生理作用。如果伴侣突变体的生长优势主要是由于替代的 20S 蛋白酶体增强了受损蛋白质的降解，那么突变体应该表现出更快的这些蛋白质降解速率。细胞中此类蛋白质的降解增加，产生替代蛋白酶体，也可能会延长按时间顺序排列的寿命。这将通过操纵相对于标准蛋白酶体的替代水平并在有或没有氧化应激的情况下长时间培养所得菌株来进行测定。由于缺乏可分离的早期组装中间体，了解这些伴侣如何指导 20S 蛋白酶体的亚基排列很难在体内研究。目标 2 提出了一种体外方法来重建 20S 蛋白酶体组装体。该方法适用于揭示 Pba3/Pba4 作用机制的各种操作。将在有或没有 Pba3/Pba4 的情况下评估 20S 蛋白酶体的组成，以确定这些伴侣发挥作用的基本方法。 相关性：许多与衰老相关的疾病，例如阿尔茨海默病和帕金森病，其特征是氧化损伤蛋白质的积累。 20S 蛋白酶体似乎在降解此类异常蛋白质方面具有特殊作用。因此，了解不同形式的 20S 蛋白酶体是如何组装的，这对这些受损蛋白质的降解效率产生重大影响，对于预防和治疗这些疾病至关重要。