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）：抑制蛋白质合成是对毒性的一般测量。进化上，虽然抑制蛋白质合成可以节省能量并防止异常的蛋白质，但越来越多的证据表明，选择性蛋白质翻译发生并确定细胞命运。已知砷和许多环境有毒物质会诱导氧化应激。我们发现，通过砷或氧化剂的培养物治疗人类细胞会导致由于从头蛋白翻译而引起的快速升高NRF2蛋白。 NRF2编码调节抗氧化剂和排毒基因网络的转录因子，在多器官系统中起到保障的作用。 NRF2敲除小鼠对砷对组织损伤的敏感性增加。了解细胞在氧化应激下如何编排导致从头NRF2蛋白翻译的分子事件对于拨打该器官保护途径很重要。人NRF2基因编码555个未翻译区（5'UTR）的555个核苷酸（NT）的mRNA物种。 5'UTR中含有内部核糖体入口位点（IRE）的几个基因可以绕过5'7- 7-甲基鸟嘌呤帽依赖性翻译，并经历应力诱导的蛋白质翻译。我们在-195至-168 NRF2 5'UTR的-195至-168核苷酸区域中发现了共识G-四链体序列。该区域的RNA片段形成了通过圆形二色性（CD），核磁共振（NMR），电泳迁移率转移测定法和硫酸二甲基硫酸二甲基脚印的G-四链体的3-D结构。基于LC-MS/MS的蛋白质组学已导致EF1A作为NRF2 5'UTR G-Quadruplex的结合伙伴的发现。在细胞水平上，氧化剂导致EF1A与NRF2 5'UTR G- Quadruplex的缔合增加，并消除了G-四倍体结构，禁止使用NRF2 5'UTR激活。 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与EIF相互作用以启动NRF2蛋白翻译。 AI 1将定义氧化应激对在细胞水平上与NRF2 5'UTR结合的蛋白质的影响。 与NRF2 5'UTR结合的蛋白质将从具有或没有氧化应激的细胞中分离出来，以通过LC-MS/MS基蛋白质组学鉴定。 AIM 2将解决EF1A与氧化应激诱导的NRF2蛋白翻译中的转化机制的相互作用。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，将解决EF1A与NRF2 5'UTR的结合是否引起转化机械的募集。 AIM 3将证实EF1A与NRF2 5'UTR相互作用在NRF2蛋白翻译，细胞存活和抗砷毒性的保护中的生物学意义。使用G Qu-Quadruplex的G-四链体适体和药理学增强剂或抑制剂，我们将测试NOVO NRF2蛋白翻译对细胞存活和小鼠组织损伤的影响。