Molecular mechanisms of ZAK- and GCN2-mediated signaling in response to ribotoxic stress

ZAK 和 GCN2 介导的信号响应核糖应激的分子机制

• 批准号: 10740532
• 负责人: Niladri K Sinha
• 金额: $ 11.61万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740532
• 关键词: Apoptosis; Atlases; Biochemical; Biological Assay; Carcinoma; Cell Cycle Arrest; Cell Line; Cell Survival; Cells; Cessation of life; Chemicals; Code; Collaborations; Cues; Data; Defect; Distress; Engineering; Equilibrium; Event; FRAP1 gene; Feedback; Gene Expression; Genetic; Goals; Growth and Development function; Homeostasis; JNK-activating protein kinase; Laboratories; Life; MAP Kinase Kinase Kinase; MAPK8 gene; Malignant - descriptor; Measurement; Measures; Mediating; Memorial Sloan-Kettering Cancer Center; Mentors; Messenger RNA; Modeling; Molecular; Monitor; Mutagens; Mutation; Outcome; Pathway interactions; Phosphotransferases; Positioning Attribute; Proteomics; RNA; Reactive Oxygen Species; Reporter; Research; Resolution; Ribosomes; Role; Signal Pathway; Signal Transduction; Site; Specificity; Stress; Stress Response Signaling; Technology; Testing; Therapeutic; Time; Training; Translating; Ultraviolet Rays; Variant; analog; biological adaptation to stress; cellular imaging; cofactor; coping; experimental study; gain of function; genetic manipulation; imaging approach; in vivo; insight; kinase inhibitor; live cell imaging; loss of function; novel; p38 Mitogen Activated Protein Kinase; phosphoproteomics; prevent; programs; response; skills; skin damage; tenure track; transcriptome; tumorigenic

Project Summary / Abstract The ability of cells to recognize and respond to extrinsic and intrinsic perturbations is critical for their survival. Recent studies have shown that the status of translating ribosomes is constantly monitored by surveillance factors to determine signs of translational distress. For example, the exposure of cells to ultraviolet radiation and reactive oxygen species causes transcriptome-wide RNA damage and prolonged stalling of ribosomes within the coding sequence of damaged messenger RNAs triggers ribosomal collisions. Recently, our laboratory has shown that the mitogen-activated protein kinase kinase kinase ZAK, and the general control nonderepressible 2 (GCN2) kinase are activated on collided ribosomes to trigger activation of the Ribotoxic and Integrated Stress Response programs (RSR and ISR) respectively. The goal of my proposal is to understand how the cell mounts a measured ZAK- and GCN2-mediated response that depends on the pervasiveness of ribosome collisions to determine cell fate. In preliminary experiments, I performed quantitative time-resolved phosphoproteomics to determine the temporal order of activation of signaling cascades in cells responding to ribotoxic stress. The data thus generated is providing an unprecedented high-resolution atlas of signaling events occurring in response to ribosome collisions and will be further explored in two proposed aims. In Aim 1, I will determine how ZAK is activated in response to ribosomal collisions and regulates cell fate outcomes through activation of the p38 and JNK signaling cascades. I will also test how a negative-feedback loop for ZAK enables cells to mount a measured response to ribotoxic stress. These studies will employ biochemical, quantitative proteomics, and live cell imaging approaches. In Aim 2, I will determine how GCN2 regulates the ISR pathway, and JNK and mTOR activity in response to ribotoxic stress by genetically and chemically perturbing GCN2’s kinase activity. Finally, I will test how co-factors and regulators of GCN2 set the threshold for its activation in response to ribosomal collisions. Together, the successful completion of these complementary approaches will provide foundational mechanistic insights into how the RSR and ISR pathways are coordinated by ZAK and GCN2 to regulate cell fate decisions during ribotoxic stress. Towards this, the experiments proposed will enable me to develop new skills and technologies in quantitative proteomics and live-cell imaging approaches, which will allow me to substantially differentiate my own research program from that of my mentor Dr. Green. I am confident that the team of collaborators that I have assembled and the accompanying training plan, will be instrumental in the growth and development of my independent research program and prepare me for a tenure-track position.

项目摘要/摘要 细胞识别和响应外在和内在扰动的能力对于它们的 生死存亡。最近的研究表明，翻译核糖体的状态一直受到 监测因素，以确定翻译困难的迹象。例如，细胞暴露在紫外线下 辐射和活性氧物种导致转录组范围的RNA损伤和长时间的停滞 受损信使RNA编码序列中的核糖体触发核糖体碰撞。最近，我们的 实验室已证明，有丝分裂原激活的蛋白激酶ZAK，与普通对照 不可降压2(GCN2)激酶在相互碰撞的核糖体上被激活，从而触发核毒素和 综合应激反应程序(RSR和ISR)分别。 我的建议的目标是了解细胞如何装载ZAK和GCN2介导的可测量的 取决于核糖体碰撞的普遍性来决定细胞命运的反应。在预赛中 实验中，我进行了定量的时间分辨磷酸蛋白质组学来确定 核毒应激引起的细胞内信号级联激活。这样生成的数据提供了一个 史无前例的响应核糖体碰撞的信号事件的高分辨率图谱，将 在两个拟议目标中进一步探讨。在目标1中，我将确定核糖体是如何激活ZAK的 通过激活p38和JNK信号通路来碰撞和调节细胞命运的结果。我也会 测试ZAK的负反馈回路如何使细胞对核毒应激做出测量的反应。 这些研究将使用生化、定量蛋白质组学和活细胞成像方法。在目标2中，我 将确定GCN2如何调节ISR途径，以及JNK和mTOR活性对核毒应激的反应 通过从遗传和化学上干扰GCN2‘S激酶活性。最后，我将测试协同因素和监管机构如何 GCN2的激活设定了响应核糖体碰撞的阈值。团结在一起，成功的 这些互补方法的完成将为RSR如何 而ISR通路由ZAK和GCN2协调，以调节核毒应激期间细胞的命运决定。 为此，提出的实验将使我能够在 定量蛋白质组学和活细胞成像方法，这将使我能够显著区分我的 我自己的研究项目来自我的导师格林博士。我相信我的合作者团队 已组装好的培训计划和随附的培训计划，将对我的成长和发展起到重要作用 独立研究计划，并为我的终身教职做好准备。