Leveraging ubiquitin-dependent regulatory mechanisms to improve proteome quality in health and disease

利用泛素依赖性调节机制提高健康和疾病中的蛋白质组质量

• 批准号: 10552479
• 负责人: Eric J Bennett
• 金额: $ 32.43万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552479
• 关键词: Aging; Biochemical; Biogenesis; Cell Line; Cell Proliferation; Cells; Cellular Stress; Chromosome abnormality; Chronic; Complex; DNA biosynthesis; Defect; Detection; Development; Disease; Dissection; Excision; Functional disorder; Genetic Transcription; Goals; Health; Human; Human Pathology; Impairment; Length; Ligase; Link; Longevity; Messenger RNA; Molecular; Mutation; Nature; Neurodegenerative Disorders; Neurologic Dysfunctions; Outcomes Research; Pathology; Pathway interactions; Persons; Prevalence; Process; Production; Proliferating; Proteins; Proteome; Quality Control; Recycling; Research; Ribosomes; Stress; Structure; System; Toxic effect; Translation Initiation; Translations; Triage; Ubiquitin; Variant; combat; fitness; genome editing; healthspan; improved; neoplastic cell; nervous system disorder; proteostasis; proteotoxicity; response; ubiquitin ligase

PROJECT SUMMARY Errors associated with DNA replication, transcription, mRNA processing, and protein biogenesis result in the continuous production of potentially toxic defective proteins. The error-prone nature of these essential processes requires robust quality control (QC) systems to effectively triage and destroy defective translation products. Protein quality control is an essential component within the larger protein homeostasis (proteostasis) system and proteostasis dysfunction has been implicated in human aging-related pathologies. On one hand, elevated protein QC function is needed to enable neoplastic cell proliferation in cells with high mutational burdens or chromosomal abnormalities. Conversely, impaired proteostasis and defects in protein QC function result in the enhanced production of misfolded and toxic aggregation prone proteins that typify many neurodegenerative disorders. These observations suggest that developing molecular strategies to predictably alter QC function to either enhance, or limit QC capacity as needed can improve aging-associated disorders and extend human healthspan. However, there is a surprising and substantial gap in our understanding of not only how QC systems selectively engage their substrates, but also how substrates evade detection during proteostasis dysfunction. To make substantive progress toward the goal of leveraging QC systems to combat aging-associated disorders, it is necessary to identify and characterize cellular and molecular mechanisms that enable detection and degradation of diverse QC substrates. Recent research progress from my lab has identified a spatially restricted QC pathway that acts on stalled and collided ribosomal complexes both before and after translation initiation to target defective translation products for degradation and recycle ribosomal complexes. Further, we have developed a systematic pipeline for biochemical, structural, and cellular interrogation of enigmatic but critical QC ubiquitin ligases that have been implicated in targeting diverse substrates for degradation by unknown mechanisms. We have focused our initial studies on the ubiquitin ligase HUWE1. Our recently described HUWE1 structure represents the first full-length structure of a HECT-domain ligase. We have generated a unique and powerful set of genome-edited cell lines and HUWE1 variants that have and will enable molecular dissection of HUWE1 function, HUWE1 substrate identification, and identification of cellular stress conditions that require HUWE1 for cellular survival and proliferation. Research outcomes achieved by the proposed studies will mechanistically determine how terminally stalled ribosomes are sensed and resolved and how ribosome-associated QC pathways can be manipulated to alter proteostasis function. Further, we will establish mechanisms by which QC ligases engage substrates under normal and stressed conditions. Successful completion of the proposed research will provide substantial progress toward our long-term goal of combating aging-associated human pathology through the development of molecular strategies to modify cellular responses to chronic proteotoxic stress and improve cellular fitness following proteostasis insults.

项目概要 与 DNA 复制、转录、mRNA 加工和蛋白质生物合成相关的错误会导致 持续生产具有潜在毒性的缺陷蛋白质。这些基本过程容易出错的性质 需要强大的质量控制 (QC) 系统来有效分类和销毁有缺陷的翻译产品。 蛋白质质量控​​制是更大的蛋白质稳态（蛋白质稳态）系统中的重要组成部分， 蛋白质稳态功能障碍与人类衰老相关的病理有关。一方面，蛋白质含量升高 需要 QC 功能来实现具有高突变负荷或染色体异常的细胞中的肿瘤细胞增殖 异常。相反，蛋白质稳态受损和蛋白质 QC 功能缺陷会导致 产生错误折叠和有毒聚集倾向的蛋白质，这些蛋白质是许多神经退行性疾病的典型特征。 这些观察结果表明，开发分子策略来可预测地改变 QC 功能 根据需要增强或限制质量控制能力可以改善与衰老相关的疾病并延长人类的健康寿命。 然而，我们不仅对 QC 系统如何选择性地进行理解，还存在令人惊讶且巨大的差距。 参与它们的底物，以及底物如何在蛋白质稳态功能障碍期间逃避检测。使 在利用质量控制系统对抗衰老相关疾病的目标方面取得了实质性进展 识别和表征能够检测和降解的细胞和分子机制所必需的 不同的 QC 底物。我的实验室最近的研究进展确定了空间受限的 QC 途径 在翻译起始至目标之前和之后作用于停滞和碰撞的核糖体复合物 用于降解和回收核糖体复合物的有缺陷的翻译产物。此外，我们还开发了一个 用于对神秘但关键的 QC 泛素进行生化、结构和细胞询问的系统化流程 连接酶涉及靶向多种底物，通过未知机制进行降解。我们 我们的初步研究集中在泛素连接酶 HUWE1 上。我们最近描述的 HUWE1 结构 代表 HECT 结构域连接酶的第一个全长结构。我们已经生成了一套独特而强大的组合 基因组编辑细胞系和 HUWE1 变体已经并将能够对 HUWE1 进行分子解剖 功能、HUWE1 底物识别以及需要 HUWE1 的细胞应激条件的识别 细胞的存活和增殖。拟议研究取得的研究成果将机械地 确定如何感测和解析最终停滞的核糖体以及如何进行核糖体相关的 QC 可以操纵途径来改变蛋白质稳态功能。此外，我们将建立QC机制 连接酶在正常和应激条件下与底物结合。顺利完成拟议的 研究将为我们对抗与衰老相关的人类的长期目标提供实质性进展 通过开发分子策略来改变细胞对慢性蛋白毒性的反应来进行病理学 压力并改善蛋白质稳态损伤后的细胞健康。