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

项目摘要 与DNA复制、转录、mRNA加工和蛋白质生物合成相关的错误导致了 持续产生潜在有毒的缺陷蛋白质。这些基本过程的易出错性 需要强大的质量控制（QC）系统来有效地筛选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（proteostasis）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学有关。一方面，高蛋白 需要QC功能，以使具有高突变负荷或染色体畸变的细胞中的肿瘤细胞增殖 异常相反，受损的蛋白质稳态和蛋白质QC功能的缺陷导致增强的蛋白质稳态。 产生错误折叠的和有毒的聚集倾向蛋白，其代表许多神经变性疾病。 这些观察结果表明，开发可预测地改变QC功能的分子策略， 根据需要增强或限制QC能力可以改善与衰老相关的疾病并延长人类健康寿命。 然而，在我们对质量控制系统如何选择性地 结合它们的底物，而且底物如何在蛋白质稳定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QC底物。我的实验室最近的研究进展已经确定了一个空间限制的QC途径 在翻译起始之前和之后作用于停滞和碰撞的核糖体复合物， 有缺陷的翻译产物用于降解和回收核糖体复合物。此外，我们还开发了一个 用于神秘但关键QC泛素的生化、结构和细胞询问的系统管道 这些连接酶涉及靶向不同的底物以通过未知机制降解。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 表示HECT结构域连接酶的第一个全长结构。我们创造了一套独特而强大的 基因组编辑的细胞系和HUWE1变体，这些变体已经并将能够对HUWE1进行分子解剖 功能，HUWE1底物鉴定，以及鉴定需要HUWE1的细胞应激条件 细胞存活和增殖。拟议研究所取得的研究成果将在机制上 确定末端停滞的核糖体是如何被感知和分辨的，以及核糖体相关的QC是如何被感知和分辨的。 可以操纵途径以改变蛋白质稳定功能。此外，我们将建立机制， 连接酶在正常和应激条件下接合底物。圆满完成拟议的 研究将为我们对抗与衰老相关的人类疾病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， 应激和改善蛋白质稳态损伤后的细胞适应性。

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.