Translation Control of Oxidative Stress

氧化应激的平移控制

• 批准号: 8899613
• 负责人: QIN M CHEN
• 金额: $ 26.78万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899613
• 关键词: 3-Dimensional; 5' Untranslated Regions; Address; Affect; Antioxidants; Arsenic; Binding; Biological; Biological Assay; Bypass; Cell Survival; Cells; Circular Dichroism; Color; Consensus; Cytoprotection; Drug Metabolic Detoxication; Electrophoretic Mobility Shift Assay; Enhancers; Eukaryotic Initiation Factors; Event; Far-Western Blotting; Figs - dietary; G-Quartets; Genes; Guanine; Health; Human; Immunoprecipitation; Injury; Knockout Mice; Mammalian Cell; Measurement; Measures; Mediating; Messenger RNA; Molecular; Mus; NF-E2-related factor 2; Nuclear Magnetic Resonance; Nucleotides; Organ; Oxidants; Oxidative Stress; Pathway interactions; Protein Binding; Protein Biosynthesis; Protein Synthesis Inhibition; Proteins; Proteomics; RNA; RNA-Protein Interaction; Regulatory Element; Resolution; Ribonucleoproteins; Ribosomes; Role; Site; Solutions; Stress; Stress-Induced Protein; Structure; Testing; Tissues; Toxic Environmental Substances; Toxic effect; Trans-Activators; Translating; Translation Initiation; Translations; Work; aptamer; base; body system; cell type; dimethyl sulfate; eIF-4B; eukaryotic initiation factor-5B; gene function; inhibitor/antagonist; oxidation; prevent; stem; transcription factor

DESCRIPTION (provided by applicant): Inhibition of protein synthesis is a general measurement of toxicity. Evolutionarily while inhibition of protein synthesis serves to save energy and prevents aberrant proteins being made, increasing evidence suggests that selective protein translation occurs and determines the cell fate. Arsenic and many environmental toxicants are known to induce oxidative stress. We found that treatment of human cells in culture with arsenic or oxidants causes rapid elevation Nrf2 protein due to de novo protein translation. Nrf2 encodes a transcription factor regulating a network of antioxidant and detoxification genes, functioning as a safeguard in multiple organ systems. Nrf2 knockout mice show an increased sensitivity to tissue injury by arsenic. Understanding how cells orchestrate molecular events leading to de novo Nrf2 protein translation under oxidative stress is important for dialing up this pathway for organ protection. Human Nrf2 gene encodes an mRNA species containing 555 nucleotides (nt) of 5' Untranslated Region (5'UTR). Several genes containing an Internal Ribosomal Entry Site (IRES) in 5'UTR can bypass 5' 7-methyl Guanine cap dependent translation and undergo stress induced protein translation. We found a consensus G-quadruplex sequence in -195 to - 168 nucleotide region of Nrf2 5'UTR. An RNA fragment from the region forms the 3-D structure of G-quadruplex as measured by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), Electrophoretic Mobility Shift Assay and Dimethyl Sulfate footprinting. LC-MS/MS based proteomics has led to the discovery of EF1a as a binding partner of Nrf2 5'UTR G-quadruplex. At the cellular level, oxidants cause an increased association of EF1a with Nrf2 5'UTR G-quadruplex and eliminating the G- quadruplex structure prohibited the activation of Nrf2 5'UTR. Since an RNA strand in cells is rarely free of protein binding, the G-quadruplex structure forms in solution from a naked RNA fragment, and oxidation of Guanine does not affect G-quadruplex formation, we hypothesize that oxidative stress causes changes in the proteins binding to Nrf2 5'UTR at the cellular level, resulting in G-quadruplex formation and recruitment of specific proteins for interaction with eIFs to initiate Nrf2 protein translation. Ai 1 will define the impact of oxidative stress on proteins binding to Nrf2 5'UTR at the cellular level. Proteins binding to Nrf2 5'UTR will be isolated from cells with or without oxidative stress for identification by LC-MS/MS based proteomics. Aim 2 will address the interplay of EF1a with translational machinery in oxidative stress induced Nrf2 protein translation. Whether EF1a binding to Nrf2 5'UTR causes recruitment of translational machinery will be addressed by examining the interaction of EF1a/Nrf2 mRNA with eIFs, ribosomes and ribosome associated proteins. Aim 3 will confirm the biological significance of EF1a interaction with Nrf2 5'UTR in Nrf2 protein translation, cell survival and protection against arsenic toxicity. Using a G-quadruplex aptamer and pharmacological enhancers or inhibitors of G-quadruplex, we will test the effect of de novo Nrf2 protein translation in cell survival and mouse tissue injury by arsenic.

描述（由申请人提供）：蛋白质合成的抑制是毒性的一般测量。从进化角度来看，虽然抑制蛋白质合成可以节省能量并防止异常蛋白质的产生，但越来越多的证据表明选择性蛋白质翻译会发生并决定细胞的命运。已知砷和许多环境毒物会诱发氧化应激。我们发现，用砷或氧化剂处理培养物中的人类细胞会由于从头蛋白质翻译而导致 Nrf2 蛋白快速升高。 Nrf2 编码一种转录因子，调节抗氧化和解毒基因网络，在多个器官系统中发挥保护作用。 Nrf2 敲除小鼠表现出对砷造成的组织损伤的敏感性增加。了解细胞如何协调分子事件，导致氧化应激下 Nrf2 蛋白从头翻译，对于调节这条器官保护途径非常重要。人类 Nrf2 基因编码含有 5' 非翻译区 (5'UTR) 555 个核苷酸 (nt) 的 mRNA 种类。 5'UTR 中包含内部核糖体进入位点 (IRES) 的多个基因可以绕过 5'7-甲基鸟嘌呤帽依赖性翻译并进行应激诱导的蛋白质翻译。我们在 Nrf2 5'UTR 的 -195 至 -168 核苷酸区域中发现了共有的 G-四链体序列。通过圆二色性 (CD)、核磁共振 (NMR)、电泳迁移率变动测定和硫酸二甲酯足迹法测量，该区域的 RNA 片段形成 G-四链体的 3-D 结构。基于 LC-MS/MS 的蛋白质组学发现 EF1a 作为 Nrf2 5'UTR G-四联体的结合伴侣。在细胞水平上，氧化剂导致 EF1a 与 Nrf2 5'UTR G-四链体的关联增加，消除 G-四链体结构会阻止 Nrf2 5'UTR 的激活。由于细胞中的RNA链很少不与蛋白质结合，G-四链体结构在溶液中由裸露的RNA片段形成，并且鸟嘌呤的氧化不影响G-四链体的形成，我们假设氧化应激导致细胞水平上与Nrf2 5'UTR结合的蛋白质发生变化，导致G-四链体形成并招募特定蛋白质 与 eIF 相互作用启动 Nrf2 蛋白翻译。 Ai 1 将在细胞水平上定义氧化应激对与 Nrf2 5'UTR 结合的蛋白质的影响。 将从有或没有氧化应激的细胞中分离出与 Nrf2 5'UTR 结合的蛋白质，以便通过基于 LC-MS/MS 的蛋白质组学进行鉴定。目标 2 将解决氧化应激诱导的 Nrf2 蛋白翻译中 EF1a 与翻译机制的相互作用。通过检查 EF1a/Nrf2 mRNA 与 eIF、核糖体和核糖体相关蛋白的相互作用，可以确定 EF1a 与 Nrf2 5'UTR 的结合是否会导致翻译机制的募集。目标 3 将确认 EF1a 与 Nrf2 5'UTR 相互作用在 Nrf2 蛋白翻译、细胞存活和砷毒性保护中的生物学意义。使用 G-四链体适体和 G-四链体的药理学增强剂或抑制剂，我们将测试从头 Nrf2 蛋白翻译对细胞存活和砷引起的小鼠组织损伤的影响。