Oxidative stress response and metabolic reprogramming by protein posttranslational arginylation

氧化应激反应与蛋白质翻译后酰基化代谢重编程 翻译后精氨酸化是在蛋白质上增加一个额外的精氨酸。这种变化往往导致 蛋白质快速降解在真菌和动物中，腺苷酸化仅由精氨酰转移酶1（ATE 1）介导。 来自我们小组和其他人的多条证据表明，ATE 1及其乙酰化活性是 对于细胞对各种氧化应激和相关代谢重编程的反应至关重要 包括糖酵解。然而，腺苷酸化调节氧化应激的分子机制 反应（OSR）和代谢仍然未知。这种知识上的差距使我们很难设计出 干预腺苷酸化以预防或治疗疾病如炎症的方法， 心血管异常，癌症和与衰老有关的疾病，这些疾病通常来自于调节失调， OSR和相关的代谢改变。 根据我们最近的研究，我们发现ATE 1和腺苷酸化活性在细胞中增加， 在急性氧化应激下，慢性暴露于应激源后下调，我们的目的是了解 乙酰基化是如何影响OSR的对乙酰基化缺乏了解主要是由于 在鉴定大多数快速降解的乙酰基化底物的领域中存在技术挑战。到 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 乙酰基化对蛋白质稳定性的影响以及鉴定蛋白质上乙酰基化修饰的直接方法。功率 在我们的初步筛选中，我们发现了几种新的 包括低氧诱导因子1 β（HIF 1 β），OSR，糖酵解， 和线粒体呼吸。我们的数据进一步表明，HIF 1a的功能作用是由以下因素调节的： 去乙酰化。继这一突破之后，这项拟议研究的目标是揭示乙酰化是如何 通过影响关键蛋白质调节氧化应激反应和相关的代谢重编程 包括HIF 1 α。我们将应用和扩展我们新开发的方法，以确定额外的乙酰化 蛋白质（Aim 1），阐明了在调节应激中对包括HIF 1 α在内的关键底物的乙酰化作用 反应/代谢（目标2），并使用高通量方法来表征全球影响 不同细胞途径上的乙酰化（Aim 3）。 在本研究的最后，我们有望揭示翻译后表达的主要分子机制， HIF 1 α和其他关键蛋白质的β-羟化调节OSR和代谢。我们还将发现全球 在许多细胞途径中，由单一酶ATE 1介导的腺苷酸化的影响。持久 通过发现许多新的和意想不到的乙酰基化靶点，可以进一步预期这种影响。

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.