Proteasome homeostasis and substrate prioritization

蛋白酶体稳态和底物优先顺序

• 批准号: 10623563
• 负责人: Jeroen Roelofs
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623563
• 关键词: Aging; Biochemical; Biological Assay; Cell physiology; Cells; Complex; Foundations; Goals; Growth; Heat Stress Disorders; Homeostasis; Human; Knowledge; Liquid substance; Malignant Neoplasms; Molecular; Neurodegenerative Disorders; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Phase; Physical condensation; Process; Proteins; Research; Saccharomyces cerevisiae; Saccharomycetales; Stress; Testing; Therapeutic; Transcriptional Regulation; Ubiquitination; Up-Regulation; Work; Yeasts; biological adaptation to stress; human disease; in vitro Assay; in vivo; insight; model organism; multicatalytic endopeptidase complex; response; tissue culture

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.

项目总结 在最佳生长阶段，细胞有足够的蛋白酶体能力来降解已标记的蛋白质。 通过泛素化进行降解。然而，为了应对压力，如热应激或氧化应激，蛋白酶体 容量进行了调整，以满足蜂窝需求。重要的是要了解细胞是如何调节这种调节的 随着年龄的增长，以及神经退行性疾病和癌症等人类疾病，许多应激状态都会出现。 根据压力的类型和主要目标，观察到了不同的细胞反应 这一建议是为了从机制上洞察应激后不同的蛋白酶体反应。这个 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 蛋白酶体复合体中新合成的亚基的组装。一种令人惊讶的复杂性存在于早期 不清楚各种蛋白酶体相关因子，如Pba1-Pba2/PAC1-PAC2， Fub1/PI31和Blm10/PA200有助于组装过程的连续步骤或平行路径。 理解这些途径是本提案的重点，因为它影响到 形成蛋白酶体，从而形成细胞的降解能力。蛋白酶体的重新定位是另一个原因。 胁迫反应，包括胞质冷凝物的形成或自噬靶向(蛋白吞噬)。 凝析油通过液-液分离形成，具有存储蛋白酶体、保护蛋白酶体的功能 或将它们与降解中心的底物一起浓缩。这样的形成 凝析油一般取决于多价相互作用的形成。推动这一点的相互作用 然而，蛋白酶体的过程还没有被很好地理解。为了对这一过程有新的见解， 拟议的研究将利用新发现的条件和因素来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， 蛋白酶体将某些底物的降解优先于其他底物。这是一部原创而又重要的 这一概念将使用体内降解试验进行分析。我们的目标是确定蛋白酶体如何- 相关蛋白质有助于这种优先顺序的确定。 蛋白酶体对压力的反应在酵母和人类细胞中都已被描述，表明 进化保守的反应，与各种细胞过程错综复杂地联系在一起，并不断变化 条件。利用发芽酵母和哺乳动物组织培养，进行了生化和细胞生物学检测 这里提出的建议将提供对决定水平、组成、 以及细胞中蛋白酶体的底物优先顺序。这样的洞察力将提供关于 这些重要的细胞复合体为努力识别靶点和增加药物创造了基础 或降低蛋白酶体水平和细胞内的局部活性。在体内操纵蛋白酶体的能力 已被证明具有治疗价值。