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.

项目总结 与DNA复制、转录、mRNA处理和蛋白质生物发生相关的错误导致 持续生产具有潜在毒性的缺陷蛋白。这些基本过程容易出错的性质 需要强大的质量控制(QC)系统来有效地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)系统中的一个重要组成部分， 蛋白平衡功能障碍已被认为与人类衰老相关的病理有关。一方面，蛋白质含量升高 QC功能是在具有高突变负荷或染色体的细胞中实现肿瘤细胞增殖所必需的 异常现象。相反，蛋白稳定性受损和蛋白QC功能缺陷会导致 产生错误折叠和有毒的聚集倾向蛋白，这是许多神经退行性疾病的典型特征。 这些观察表明，开发分子策略来可预测地改变QC功能 根据需要加强或限制QC能力可以改善与衰老相关的疾病，延长人类的健康寿命。 然而，在我们对QC系统如何选择性地理解方面存在着令人惊讶的和实质性的差距 接触底物，以及底物如何在蛋白代谢功能障碍期间逃避检测。使 在实现利用QC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 不同的质量控制基材。我的实验室最近的研究进展确定了一条空间受限的QC途径 在靶向翻译起始前后作用于停滞和碰撞的核糖体复合体 有缺陷的翻译产物，用于降解和回收核糖体复合体。此外，我们还开发了一种 用于神秘但关键的QC泛素的生化、结构和细胞询问的系统流水线 连接酶被认为是以不同的底物为目标进行降解的未知机制。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。我们已经产生了一套独特而强大的 基因组编辑的细胞系和HUWE1变异体具有并将使HUWE1分子解剖成为可能 功能、HUWE1底物鉴定和需要HUWE1的细胞应力条件的鉴定 细胞的存活和增殖。拟议研究取得的研究成果将机械地 确定末端停滞的核糖体是如何被感知和分解的，以及核糖体相关的质量控制是如何 可以通过操纵通路来改变蛋白平衡功能。此外，我们将建立QC 连接酶在正常和应激条件下与底物结合。圆满完成拟议中的 研究将为我们抗击与衰老相关的人类的长期目标提供实质性进展 通过发展分子策略来改变细胞对慢性蛋白毒性的反应的病理学 应激并改善蛋白质平衡侮辱后的细胞健康状况。