Translational control of ROS Management

描述(由申请人提供)：环境和内源过程都会产生病理性的高水平ROS，随后对脂质、蛋白质和DNA的损害会促进突变和细胞死亡。然而，低水平的ROS在调节细胞增殖和基因表达方面具有重要的生理意义。因此，1，2细胞必须防止ROS的有害方面，并促进ROS的有益方面，这表明有精确的调控策略来维持适当的ROS水平。我们建议利用RNA甲基转移酶ALKBH8和TRM9L相反的活性，通过打开和关闭ROS解毒和DNA损伤反应蛋白的翻译来检验细胞通过打开和关闭ROS解毒和DNA损伤反应蛋白的翻译来管理ROS平衡的假设。其潜在机制源于我们的发现，即ALKBH8酶催化的甲基化信号，在tRNA修饰反密码子的水平上，可以翻译上调含硒半胱氨酸(SEC)的ROS管理蛋白。这些RNA修饰是“终止密码子重新编码”所必需的--即在含有SEC的谷胱甘肽过氧化物酶(GPX，抗氧化剂)和硫氧还蛋白还原酶(TrxRs，调节核糖核苷酸还原酶活性以促进DNA损伤反应)的mRNAs中，将内部终止密码子读作SEC密码子的机制。我们假设，决斗的RNA修饰控制停止密码子重新编码，以促进(ALKBH8)或阻止(TRM9L)SEC-蛋白质的翻译，从而调节ROS和DNA损伤反应。此外，我们假设致癌过程的一部分涉及劫持RNA修饰系统以关闭应激反应，并促进ROS诱导的DNA损伤和DNA修复减少的亲突变程序。为了便于我们的研究，我们开发了一个修饰分析平台来监测33个人类tRNA修饰，我们已经证明了反密码子修饰在ROS应激反应中受到显著调控。与我们的RNA修饰分析平台一起，我们将使用生化、分子和计算分析在一组健壮的正常、癌症衍生和可工程的品系中测试我们的假设。重要的是，我们的研究将定义一种新的应激反应翻译控制系统，剖析促突变程序的机制，并揭示癌症治疗和预防的新靶点和新策略。

DESCRIPTION (provided by applicant): Both environmental and endogenous processes generate pathologically high levels of ROS, with subsequent damage to lipids, proteins and DNA promoting mutation and cell death. However, low levels of ROS are physiologically important in regulating cell proliferation and gene expression.1,2 Cells must thus prevent the detrimental and promote the beneficial aspects of ROS, which suggests that there are precise regulatory strategies to maintain proper ROS levels. We propose to test the hypothesis that cells manage ROS balance by turning-on and turning-off the translation of ROS detoxification and DNA damage response proteins, using the opposing activities of the RNA methyltransferases ALKBH8 and TRM9L. The underlying mechanism arises from our discovery that enzyme-catalyzed methylation signals by ALKBH8, operating at the level of tRNA modification to anticodons, can translationally up-regulate selenocysteine (Sec)-containing ROS management proteins. These RNA modifications are required for "stop-codon recoding" - the mechanism of reading internal stop-codons as Sec-codons in mRNAs for Sec-containing glutathione peroxidases (GPXs, antioxidants) and thioredoxin reductases (TrxRs, regulate ribonucleotide reductase activity to promote the DNA damage response). We hypothesize that dueling RNA modifications control stop-codon recoding to promote (ALKBH8) or prevent (TRM9L) the translation of Sec-proteins and thus regulate ROS and DNA damage responses. Further, we hypothesize that part of the process of carcinogenicity involves hijacking of RNA modification systems to turn-off stress responses and promote a pro-mutagenic program of ROS-induced DNA damage and decreased DNA repair. To facilitate our studies, we have developed a modification analysis platform to monitor 33 human tRNA modifications and we have demonstrated that anticodon modifications are significantly regulated in response to ROS stress. Together with our RNA modification analysis platform, we will use biochemical, molecular and computational analyses to test our hypothesis in a robust set of normal, cancer- derived and engineerable lines. Importantly, our studies will define a novel system of translational control of stress response, dissect the mechanism a pro-mutagenic program, and reveal new targets and strategies for cancer treatment and prevention.