Defining Novel Roles of Ubiquitin Accumulation During the Mammalian Oxidative Stress Response

摘要 人类经历了许多有害的环境暴露，如紫外线辐射、空气污染物和暴露 对于砷化合物，通过在体内积累活性氧物种(ROS)而导致氧化应激 手机。大量的疾病进展是由氧化应激引起的，如癌症、神经退行性变和 动脉粥样硬化和慢性肾脏疾病。为了应对氧化应激，人类重新连接了细胞过程 在转录、翻译和翻译后层面上。这一回应的一个中心方面包括 泛素修饰是细胞生存所必需的，因为它在氧化应激过程中的整体积累。这些角色 促进细胞防御包括蛋白酶体降解、自噬、翻译调控和抗氧化剂 蛋白质生产。然而，泛素化在氧化应激背景下的许多作用仍然存在 未被开发的。我的赞助商的实验室已经开始发现氧化应激下泛素化的新作用， 例如，阻止特定K63泛素连接的突变如何增加酵母对氧化应激的敏感性。 此外，最近未发表的发现突出了K63泛素在内质网中的积聚。 (Er)在哺乳动物细胞的氧化应激下，这可能会改变核糖体。这项建议旨在 在我的第一篇文章中，描述了泛素修饰在人类氧化应激反应中的重要作用 目的是通过定义这种本地化K63积累的结果，在我的第二个目标中，通过定义以下关键角色 通过使用CRISPR功能丧失筛查，泛素酶在细胞活力中的应用。为了目标一号，我会 首先测试DUB活性、自噬和蛋白酶体降解对去除ER K63泛素的影响 免疫印迹和免疫荧光检测应激恢复。如果与配音活动有关，这是我的主导 假设，然后我将使用siRNA从候选列表中识别其耗尽的特定内质网驻留Dub 阻止恢复，然后通过体外和体内研究确定氧化还原水平如何调节DUBS。为 目标二，我将使用sgRNA文库针对40/40个E2、512/~600个E3和90/102个Dub来识别候选 在较高的ROS水平下sgRNAs表达不足的酶。一旦确定，我将定义 最不具代表性的酶通过确定其与应激相关的泛素化靶标的作用，如 以及氧化如何调节其功能。更深入地了解泛素在血管紧张素转换酶的基本机制中的作用 氧化应激反应途径将为临床策略提供信息，以减缓ROS下的疾病进展 积累。这项提议最终将支持我成为一名独立的内科科学家。 我的培训计划包括在多个会议上展示我的这项工作的发现，以及有组织的互动 和我的指导团队一起。在F30的支持下，我将培养成功过渡所需的技能 参加我的博士后和住院医师培训。

ABSTRACT Many harmful environmental exposures humans experience, such as UV radiation, air pollutants, and exposure to arsenic compounds, lead to oxidative stress from the accumulation of reactive oxygen species (ROS) in the cell. A vast array of disease progression results from oxidative stress, such as cancer, neurodegeneration, and atherosclerosis, and chronic kidney disease. In response to oxidative stress, humans rewire cellular processes at the transcriptional, translational, and post-translational level. A central aspect of this response includes the ubiquitin modification, as its global accumulation during oxidative stress is essential for cell survival. These roles that promote cellular defense include proteasome degradation, autophagy, translational control, and antioxidant protein production. However, many roles of the ubiquitination in the context of oxidative stress remain unexplored. My sponsor’s laboratory has begun to uncover new roles for ubiquitination under oxidative stress, such as how mutations preventing specific K63 ubiquitin linkages increase sensitivity to oxidative stress in yeast. Additionally, recent unpublished findings highlight an accumulation of K63 ubiquitin at the endoplasmic reticulum (ER) under oxidative stress in mammalian cells, which is likely to modify ribosomes. This proposal aims to characterize vital roles of the ubiquitin modification within the oxidative stress response in humans, in my first aim by defining the outcome of this localized K63 accumulation, and in my second aim by defining vital roles for ubiquitin enzymes in cell viability through use of a pooled CRISPR loss-of-function screen. For aim one, I will first test the impact of DUB activity, autophagy and proteasome degradation on removing ER K63 ubiquitin during stress recovery by western blot and immunofluorescence. If related to DUB activity, which is my leading hypothesis, I will then use siRNAs to identify a specific ER-resident DUB from a candidate list whose depletion prevents recovery, then determine how redox levels regulates the DUBs through in vitro and in vivo studies. For aim two, I will use sgRNA libraries to target 40/40 E2s, 512/~600 E3s, and 90/102 DUBs to identify candidate enzymes whose sgRNAs become underrepresented under higher ROS levels. Once identified, I will then define the roles of the most underrepresented enzyme by identifying its stress-associated targets of ubiquitination, as well as how oxidation regulates its function. Deeper understanding of ubiquitin’s role in the basic mechanisms of oxidative stress response pathways will inform clinical strategies to mitigate disease progression under ROS accumulation. This proposal will ultimately support my training to become an independent physician-scientist. My training plan includes presenting my findings of this work at multiple conferences, and structured interactions with my mentoring team. With the support of this F30, I will develop the required skill set to successfully transition to my post-doctoral and residency training.