Redox regulation of the UPR sensor Ire1

项目摘要/摘要 内质网(ER)中的氧化蛋白质折叠机制目前被认为是 潜在的重要来源的活性氧类(ROS)。然而，几乎没有具体的机制被 描述了细胞如何在内质网管理ROS的产生和/或适应ER RO的积累。使用 对细胞内ER ROS管理的途径以及ER ROS水平的异常调节知之甚少 对疾病的影响还没有得到充分的研究。这项建议的研究目标是阐明细胞如何使用ROS 在内质网管腔中产生，以启动信号，促进健康的ROS水平，并通过 氧化内质网应激的条件。为了剖析内质网产生的特定分子机制 ROS，Sevier实验室利用保守酶Ero1(Ero1*)的突变体作为一种创新方式 在酿酒酵母的内质网管腔内诱导一团过氧化氢。使用Ero1*在内质网内诱导RO，即 发现了UPR传感器IRE1在感应ER ROS中的作用。在线虫中发现了一种氧化还原 在可以被细胞质ROS激活的IRE-1的激动域中切换，我已经建立了 在保守的半胱氨酸(IRE1-C832S)上通过基因阻断IRE1氧化导致IRE1酶活性增强 在Ero1*处理期间的活性，与C832氧化导致的UPR抑制一致。基于 这些数据，我假设，在蛋白质折叠过程中，内质网管腔中产生的ROS可以氧化IRE1 C832在 细胞质并启动抗氧化反应，使细胞适应过量内质网的独特压力 罗斯。为了验证这一假设，在目标1中，我将确定当Ero1*过表达时IRE1的氧化状态 在内质网管腔中产生过氧化氢积聚，我将证明IRE1修饰发生在C832。另外， 我将确定是过氧化氢从内质网腔到胞浆的运动促进了IRE1的修饰。 在目标2中，我将评估规范的IRE1激活的丧失是否与在 抗氧化剂信号。我提议的研究项目与确保成功的更广泛的培训计划相结合 当我努力成为一名研究小组组长的时候。我认为辅导是我想要的一项复杂的专业技能 以求提高。我的赞助商和我已经概述了一项培训计划，该计划将加强我的技术和 沟通技巧，也磨练了我作为导师的熟练程度。具体地说，我提出了一个计划，将 在本科生指导下描述IRE1氧化的生化效应(S) 这将完成对IRE1氧化对IRE1激酶和核糖核酸酶活性的影响的直接分析。 我相信我的个人赞助商的支持是在康奈尔大学令人兴奋的智力环境中实现的 大学生物化学、分子和细胞生物学研究生课程为我个人科学和 职业上的成功。

Project Summary/Abstract The oxidative protein folding machinery in the endoplasmic reticulum (ER) is currently appreciated as a potentially significant source of reactive oxygen species (ROS). Yet, few specific mechanisms have been described as to how cells manage ROS production at the ER and/or adapt to an accumulation of ER ROS. With little understanding of the pathways for ER ROS management in cells, how dysregulation of ER ROS levels contributes to disease is under-explored. The research goal of this proposal is to elucidate how cells use ROS generated in the ER lumen to initiate signals that promote healthy ROS levels and allow survival through conditions of oxidative ER stress. In order to dissect the molecular mechanisms specific to the production of ER ROS, the Sevier Lab takes advantage of a mutant of the conserved enzyme Ero1 (Ero1*) as an innovative way to induce a bolus of H2O2 within the ER lumen of S. cerevisiae. Using Ero1* to induce ROS within the ER, I uncovered a role for the UPR sensor Ire1 in sensing ER ROS. Guided by the discovery in C. elegans of a redox switch in the kinase domain of IRE-1 that can be activated by cytoplasmic ROS, I have established that genetically blocking Ire1 oxidation at the conserved cysteine (Ire1-C832S) results in enhanced Ire1 enzymatic activity during Ero1* treatment, consistent with UPR dampening as a consequence of C832 oxidation. Based on these data, I hypothesize that ROS generated in the ER lumen during protein folding can oxidize Ire1 C832 in the cytoplasm and initiate an antioxidant response that allows cells to adapt to the unique stress of excess ER ROS. To test this hypothesis, in Aim 1 I will determine the oxidation status of Ire1 when Ero1* over-expression generates a buildup of H2O2 in the ER lumen, and I will show that Ire1 modification occurs at C832. Additionally, I will establish that it is the movement of H2O2 from the ER lumen into the cytosol that facilitates Ire1 modification. In Aim 2, I will assess whether the loss in canonical Ire1 activation is associated with a gain of a new function in antioxidant signaling. My proposed research project is coupled to a broader training plan that will ensure success as I work toward becoming a research group leader. I identified mentoring as a complex professional skill I want to improve. My Sponsors and I have outlined a training plan that will both strengthen my technical and communication skills and also hone my proficiency as a mentor. Specifically, I put forward a plan to integrate the characterization of the biochemical effect(s) of Ire1 oxidation with the mentoring of an undergraduate student that will complete a straightforward analysis of the impact for Ire1 oxidation on Ire1 kinase and RNase activities. I believe the support of my individual Sponsors nestled within the intellectually exciting environment of the Cornell University Biochemistry, Molecular, and Cell Biology graduate program primes me for personal scientific and professional successes.