Defining the function and mechanism of regulatory ribosomal ubiquitylation

项目总结 蛋白质稳态依赖于对有缺陷的翻译的持续监测和去除 与信使核糖核酸翻译相关的错误率相对较高的产品。蛋白平衡 功能障碍与人类衰老相关的病理有关，包括许多神经退行性疾病。 紊乱，这表明分子策略要么限制错误翻译产物的产生 或提高蛋白质质量控制能力可能会提供治疗益处。因此，将蜂窝 需要调节翻译活动或核糖体相关质量控制功能的机制来 在正常和应激条件下，使分子能够控制蛋白质的稳定。我们发现 个体40S核糖体上保守的、位点特异的、调节性的核糖体泛素化(RRub)事件 代表新的翻译控制轴的蛋白质。我们的目标是确定分子 RRub影响核糖体相关质量控制和综合应激反应的机制 路径。为了实现这一目标，我们已经确定了关键的泛素连接酶和脱泛素化酶 调解这些RRub事件。我们已经产生了一组独特而强大的基因组编辑细胞系， 将使RRub和需要RRub才能正常发挥功能的细胞通路的分子解剖成为可能。 我们的假设是，RRub机器的操作可以用于在和期间改变翻译 在急性蛋白毒性应激之后。此外，我们假设翻译活性升高的细胞 和/或受损或切割的mRNAs水平升高将需要增强功能的质量控制活动 和生存。为了探索这些假设，我们将：(1)剖析泛素依赖和独立 核糖体相关质量控制途径内的机制；(2)确定生理相关 需要提高RQC活性的细胞条件；以及(3)描述RRub如何在 稳态以及在综合应力响应的激活和恢复期间。研究成果 所提出的研究成果将从机械上决定如何感知终末停滞的核糖体。 并通过RQC途径解决。我们还将定义RRub如何通过 核糖体丰度或翻译活性的调节。几种核糖体蛋白与翻译相关 因子是调节泛素化的靶点，这表明我们的研究策略可以广泛应用。 到其他目标，以实现在多个步骤中的蛋白质生物发生控制。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。

PROJECT SUMMARY Protein homeostasis (proteostasis) relies on the continual surveillance and removal of defective translation products resulting from the relatively high error rates associated with mRNA translation. Proteostasis dysfunction has been implicated in human aging-related pathologies, including many neurodegenerative disorders, suggesting that molecular strategies to either limit the production of erroneous translation products or elevate protein quality control capacity may provide therapeutic benefit. As such, characterizing cellular mechanisms that regulate translation activity or ribosome-associated quality control function is needed to enable molecular control over proteostasis under normal and stress conditions. We have discovered conserved, site-specific, regulatory ribosomal ubiquitylation (RRub) events on individual 40S ribosomal proteins that represent a new axis of translational control. Our objective is to determine the molecular mechanisms by which RRub impacts ribosome-associated quality control and the integrated stress response pathway. Toward this goal, we have identified the critical ubiquitin ligases and deubiquitylating enzymes that mediate these RRub events. We have generated a unique and powerful set of genome-edited cell lines that will enable molecular dissection of RRub and the cellular pathways which require RRub for proper function. Our hypothesis is that manipulation of RRub machinery can be utilized to alter translation both during and following acute proteotoxic stress. Furthermore, we hypothesize that cells with elevated translation activity and/or elevated levels of damaged or cleaved mRNAs will require enhanced quality control activity for function and survival. To probe these hypotheses, we will: (1) dissect ubiquitin-dependent and independent mechanisms within the ribosome-associated quality control pathway; (2) determine physiologically-relevant cellular conditions that require elevated RQC activity; and (3) characterize how RRub reshapes translation at steady-state and during activation and recovery of the integrated stress response. Research outcomes achieved by the proposed studies will mechanistically determine how terminally stalled ribosomes are sensed and resolved via the RQC pathway. We will also define how RRub alters stress response pathways through regulation of ribosome abundance or translation activity. Several ribosomal proteins and translation-associated factors are regulatory ubiquitylation targets which suggests that our research strategy can be broadly applied to other targets to enable protein biogenesis control at multiple steps. 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.