Oxidative stress response and metabolic reprogramming by protein posttranslational arginylation

蛋白质翻译后精氨酸化的氧化应激反应和代谢重编程 翻译后精氨酸化是在蛋白质中增加一个额外的精氨酸。这种修改通常会导致 蛋白质迅速降解。在真菌和动物中，精氨酸化仅由精氨酰转移酶1(ATE1)介导。 来自我们小组和其他人的多条证据表明，ATE1及其精氨酸化活性是 对细胞对各种氧化应激源的反应和相关的代谢重编程是必不可少的 包括糖酵解。然而，精氨酸化调节氧化应激的分子机制 反应(OSR)和新陈代谢仍不清楚。这种知识的鸿沟使人很难设计出 干预精氨酸化以预防或治疗炎症等疾病的方法， 心血管异常、癌症和与衰老相关的疾病，这些通常是由调节失调引起的 OSR及其相关的代谢改变。 根据我们最近的研究，我们发现ATE1和精氨酸化活性在细胞中增加 在急性氧化应激下，并在长期暴露于应激源后下调，我们的目标是了解 精氨酸化是如何影响OSR的。对精氨酸化缺乏了解在很大程度上是由于 在鉴定大多数精氨酸化底物方面的技术挑战，这些底物迅速降解。至 为了克服这个问题，我们重新设计了一种新的方法，结合间接方法来确定影响 精氨酸化对蛋白质稳定性的影响及直接鉴定蛋白质精氨酸化修饰的方法。那股力量 在我们的初步筛选中，我们发现了几个新的 精氨酸化候选基因包括缺氧诱导因子1(HIF1)，它是OSR、糖酵解、 和线粒体呼吸作用。我们的数据进一步表明，HIF1a的功能作用受 精氨酸化。根据这一突破，这项拟议的研究的目标是揭示精氨酸化如何 通过影响关键蛋白质来调节氧化应激反应和相关的代谢重编程 包括HIF1。我们将应用和扩展我们新开发的方法来识别更多的精氨酸化 蛋白质(AIM1)，阐明包括HIF1在内的关键底物精氨酸化在调节应激中的作用 响应/新陈代谢(目标2)，并使用高通量方法来表征全球影响 不同细胞途径上的精氨酸化(目标3)。 在这项研究的最后，我们有望揭示翻译后的主要分子机制 HIF1、和其他关键蛋白的精氨酸化调节OSR和新陈代谢。我们还会发现全球 精氨酸化的影响，这是由一个单一的酶ATE1，在许多细胞途径。一种持久的 许多新的和意想不到的精氨酸化靶点的发现进一步预测了影响。

Oxidative stress response and metabolic reprogramming by protein posttranslational arginylation Posttranslational arginylation is the addition of one extra arginine to a protein. This modification often leads to rapid protein degradation. In fungi and animals, arginylation is solely mediated by Arginyltransferase1 (ATE1). Multiple lines of evidence from our group and others have shown that ATE1 and its arginylation activity are essential for cellular response to a variety of oxidative stressors and the associated metabolic reprogramming including glycolysis. However, the molecular mechanisms by which arginylation regulate oxidative stress response (OSR) and metabolism remain unknown. This gap of knowledge makes it difficult to devise approaches to intervene in arginylation for the prevention or treatment of diseases such as inflammation, cardiovascular abnormalities, cancer, and aging-related maladies, which are often derived from dysregulated OSR and the associated metabolic alterations. Following from our recent studies where we showed that ATE1 and arginylation activity are increased in cells under acute oxidative stress, and downregulated upon chronic exposures to stressors, we aim to understand exactly how arginylation influences OSR. The lack of understanding for arginylation is largely due to major technical challenges in the field for identifying the majority of arginylation substrates, which degrade rapidly. To overcome this problem, we redesign a new approach combining indirect methods to identify the impact of arginylation on protein stability and direct methods to identify arginylation modification on proteins. The power of this new approach was demonstrated in our preliminary screening, in which we identified several new arginylation candidates including hypoxia-inducible factor 1 (HIF1), a critical regulator of OSR, glycolysis, and mitochondrial respiration. Our data further suggested that the functional role of HIF1a is regulated by arginylation. Following from this breakthrough, the objective of this proposed study is to reveal how arginylation regulates oxidative stress response and associated metabolic reprogramming by affecting critical proteins including HIF1. We will apply and expand our newly developed approach to identify additional arginylated proteins (Aim1), elucidate the effects of arginylation on key substrates including HIF1 in regulating stress response/metabolism (Aim 2), and using high-throughput methods to characterize the global impact of arginylation on different cellular pathways (Aim 3). In the end of this study, we are expected to reveal major molecular mechanisms of how posttranslational arginylation of HIF1 and other critical proteins regulates OSR and metabolism. We would also uncover global impacts of arginylation, which is mediated by a single enzyme ATE1, in many cellular pathways. A long-lasting impact is further anticipated by the discoveries of many new and unexpected targets of arginylation.