Translation Control of Oxidative Stress

描述（由申请人提供）：抑制蛋白质合成是毒性的一般测量。从进化的角度来看，抑制蛋白质合成是为了节省能量和防止异常蛋白质的产生，越来越多的证据表明，选择性蛋白质翻译的发生决定了细胞的命运。已知砷和许多环境毒物可诱发氧化应激。我们发现，用砷或氧化剂处理培养的人类细胞，由于从头蛋白翻译，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，在多器官系统中起保护作用。Nrf2基因敲除小鼠对砷组织损伤的敏感性增加。了解细胞如何协调导致氧化应激下Nrf2蛋白从头翻译的分子事件，对于打通这条器官保护途径非常重要。人类Nrf2基因编码一个含有555个核苷酸（nt）的5′非翻译区（5′utr）的mRNA物种。在5' utr中含有一个内部核糖体进入位点（IRES）的一些基因可以绕过5' 7-甲基鸟嘌呤帽依赖的翻译而进行应激诱导的蛋白质翻译。我们在Nrf2 5'UTR -195 ~ - 168核苷酸区发现了一致的g -四重体序列。通过圆二色性（CD）、核磁共振（NMR）、电泳迁移率转移测定和硫酸二甲基足迹测定，该区域的RNA片段形成g -四重体的三维结构。基于LC-MS/MS的蛋白质组学发现EF1a是Nrf2 5'UTR g -四重体的结合伙伴。在细胞水平上，氧化剂导致EF1a与Nrf2 5'UTR G-四重体的关联增加，消除G-四重体结构禁止Nrf2 5'UTR的激活。由于细胞中的RNA链很少不受蛋白质结合的影响，在溶液中，裸露的RNA片段形成g -四重体结构，而鸟嘌呤的氧化不影响g -四重体的形成，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 -四重体的形成，并招募特异性蛋白质与eif相互作用，启动Nrf2蛋白的翻译。Ai 1将定义氧化应激在细胞水平上对Nrf2 5'UTR结合蛋白的影响。

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.