Translation Control of Oxidative Stress

氧化应激的平移控制

• 批准号: 8747124
• 负责人: QIN M CHEN
• 金额: $ 26.69万
• 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/8747124
• 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(申请人提供)：抑制蛋白质合成是毒性的一般测量。在进化上，虽然抑制蛋白质合成有助于节省能量并防止产生异常蛋白质，但越来越多的证据表明，选择性蛋白质翻译发生并决定细胞的命运。众所周知，砷和许多环境毒物会引起氧化应激。我们发现，在用砷或氧化剂处理培养的人类细胞时，由于从头蛋白的翻译，Nrf2蛋白迅速升高。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基因敲除小鼠对砷的组织损伤表现出更高的敏感性。了解细胞如何在氧化应激下协调导致从头Nrf2蛋白翻译的分子事件，对于启动这一途径以保护器官非常重要。人类Nrf2基因编码555个核苷酸的5‘非翻译区(5’UTR)。在5‘端非编码区含有内部核糖体进入位点(IRES)的几个基因可以绕过5’7-甲基鸟嘌呤帽子依赖的翻译，进行应激诱导的蛋白质翻译。我们在Nrf2 5‘非编码区的-195~-168核苷酸区域发现了一个一致的G-四链序列。通过圆二色谱(CD)、核磁共振(核磁共振)、凝胶迁移率位移分析和硫酸二甲酯足迹测定，该区域的RNA片段形成了G-四链体的三维结构。基于LC-MS/MS的蛋白质组学导致发现EF1a作为Nrf2 5‘UTRG-四链的结合伙伴。在细胞水平上，氧化剂导致EF1a与Nrf2 5‘UTRG-四链结合增加，消除G-四链结构抑制了Nrf2 5’UTR的激活。由于细胞中的RNA链很少没有蛋白质结合，G-四链体结构在溶液中由裸露的RNA片段形成，而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-四链体的形成和特定蛋白质的募集，以便与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。 与Nrf2 5‘非编码区结合的蛋白质将从有或无氧化应激的细胞中分离出来，用于基于LC-MS/MS的蛋白质组学鉴定。目的2将探讨EF1a与翻译机制在氧化应激诱导的Nrf2蛋白翻译中的相互作用。EF1a与Nrf2 5‘UTR结合是否会导致翻译机制的招募，将通过研究EF1a/Nrf2 mRNA与EIF、核糖体和核糖体相关蛋白的相互作用来解决。目的3证实EF1a与Nrf2 5‘端非编码区相互作用在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-四链适配子和G-四链的药理增强剂或抑制剂，我们将测试从头Nrf2蛋白翻译在砷对细胞存活和小鼠组织损伤中的作用。