Oxidative stress response and metabolic reprogramming by protein posttranslational arginylation

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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.