Regulation of Alternative 20S Proteasome Assembly under oxidative stress

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26 S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具有主要作用。值得注意的是，细胞衰老被认为是由氧化损伤蛋白质的积累加速的。20 S蛋白酶体由14个不同亚基组装，由蛋白酶体生物发生相关因子3和4（Pba 3/Pba 4）指导，这有助于指导某些亚基放置到特定位置;没有这些分子伴侣，20 S蛋白酶体的外环被重塑成另一种排列。缺乏Pba 3/Pba 4的突变细胞在已知引起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20 S蛋白酶体对受损蛋白的降解增强，则突变体应表现出这些蛋白的更快降解速率。这些蛋白质在细胞中的降解增加，产生替代蛋白酶体，也可能增加时间寿命。这将通过操纵相对于标准蛋白酶体的替代物水平并在长时间内在有或没有氧化应激的情况下生长所得菌株来测定。了解这些分子伴侣如何指导20 S蛋白酶体的亚基排列是很难在体内研究的，因为缺乏可以分离的早期组装中间体。目的2提出一种体外重组20 S蛋白酶体的方法。这种方法适用于各种操作，可以揭示Pba 3/Pba 4的作用机制。将在存在和不存在Pba 3/Pba 4的情况下评估20 S蛋白酶体的组成，以确定这些分子伴侣发挥功能的基本方法。 相关性：许多与衰老有关的疾病，如阿尔茨海默病和帕金森病，其特征在于氧化损伤蛋白质的积累。20 S蛋白酶体似乎在降解这种异常蛋白质种类中具有特殊作用。因此，了解不同形式的20 S蛋白酶体是如何组装的，这可能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。

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.