Proteasome homeostasis and substrate prioritization

项目摘要 在最佳生长期间，细胞具有足够的蛋白酶体能力来降解已标记的蛋白质。 用于泛素化降解。然而，在应激反应中，如热应激或氧化应激，蛋白酶体 调整容量以满足蜂窝需求。重要的是要了解细胞如何调节这种调整， 许多应激状态发生在衰老和人类疾病中，如神经变性疾病和癌症。 已经观察到不同的细胞反应，这取决于压力的类型，以及 该建议是为了获得对应激后不同蛋白酶体反应的机制性洞察。的 蛋白酶体的上调就是这样一种反应，它不仅涉及转录调控，而且还涉及 在蛋白酶体复合物中新合成的亚基的装配。一个令人惊讶的复杂性存在于早期 组装步骤，其中不清楚各种蛋白酶体相关因子，如Pba1-Pba2/PAC1-PAC2， Fub1/PI31和Blm10/PA200有助于组装过程的顺序步骤或平行途径。 了解这些途径是本提案的重点，因为它影响了 蛋白酶体的形成，从而细胞的降解能力。蛋白酶体的重新定位是另一个 应激反应，其涉及胞质凝聚物形成或自噬靶向（蛋白吞噬）。 通过液-液相分离发生冷凝物形成，其功能是储存蛋白酶体，保护它们， 或将它们与底物一起集中在降解中心。形成这种 缩合物的形成通常取决于多价相互作用的形成。推动这一进程的相互作用 然而，蛋白酶体的过程还没有很好地理解。为了获得对该过程的新见解， 拟议的研究将利用新确定的条件和因素，调节蛋白酶体缩合物， 酵母最后，该提案将检验以下假设：在蛋白酶体能力有限的条件下， 蛋白酶体优先降解某些底物。这是一个原始的和重要的 将使用体内降解试验分析这一概念。目的是确定蛋白酶体- 相关蛋白质有助于这种优先化。 蛋白酶体对应激的反应已经在酵母和人类细胞中被描述，表明 进化保守的反应，错综复杂地连接到各种细胞过程和变化 条件利用芽殖酵母和哺乳动物组织培养， 这里提出的将提供原始和新的见解的过程，确定水平，组成， 以及细胞中蛋白酶体的底物优先化。这些见解将提供关于以下方面的基本知识： 这些重要的细胞复合体，并为确定靶点和药物的努力奠定基础， 或降低细胞中的蛋白酶体水平和局部活性。体内操纵蛋白酶体的能力 被证明具有治疗价值。

PROJECT SUMMARY During optimal growth, cells have sufficient proteasome capacity to degrade proteins that have been marked for degradation by ubiquitination. However, in response to stress, like heat stress or oxidative stress, proteasome capacity is adjusted to meet cellular demand. It is important to understand how cells regulate this adjustment as many stress conditions occur upon aging and in human diseases, like neurodegenerative diseases and cancer. Diverse cellular responses have been observed depending on the type of stress, and the overarching goal of this proposal is to gain mechanistic insight into different proteasome responses following stress. The upregulation of proteasomes is one such response, which not only involves transcriptional regulation, but also the assembly of newly synthesized subunits in proteasome complexes. A surprising complexity exists in early steps of assembly where it is unclear how various proteasome associated factors, like Pba1-Pba2/PAC1-PAC2, Fub1/PI31, and Blm10/PA200, contribute to sequential steps or parallel pathways of the assembly process. Understanding these pathways is a focus of this proposal because it impacts the amount and type of proteasomes that form and thereby the cells' degradative capacity. The relocalization of proteasomes is another stress response which involves either cytosolic condensate formation or autophagic targeting (proteaphagy). Condensate formation occurs via liquid-liquid phase separation and functions to store proteasomes, protect them from proteaphagy, or concentrate them with substrates in degradation centers. The formation of such condensates generally depends on the formation of multivalent interactions. The interactions that drive this process for the proteasome, however, are not well understood. To gain new insights into the process, the proposed research will utilize newly identified conditions and factors that regulate proteasome condensates in yeast. Finally, the proposal will test the hypothesis that under conditions of limited proteasome capacity, proteasomes prioritize the degradation of certain substrates over others. This is an original and important concept that will be analyzed using in vivo degradation assays. The goal is to determine how proteasome- associated proteins contribute to this prioritization. The proteasome responses to stress have been described in yeast as well as human cells, indicating evolutionary conserved responses that are intricately connected to various cellular processes and changing conditions. Using both budding yeast and mammalian tissue culture, the biochemical and cell biological assays proposed here will provide original and new insights into the processes that determine the levels, composition, and substrate prioritization of proteasomes in the cell. Such insights will provide fundamental knowledge about these important cellular complexes and create a foundation for efforts to identify targets and drugs that increase or decrease proteasome levels and localized activity in cells. The ability to manipulate proteasome in vivo has been shown to be of therapeutic value.