Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis

Background Nutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2 alpha O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear. Methods We used mass spectrometry to study the relationship between O-GlcNAcylation and Arg availability in breast cancer BT-549 cells. We validated eIF2 alpha O-GlcNAcylation through site-specific mutagenesis and azido sugar N-azidoacetylglucosamine-tetraacylated labeling. We then evaluated the effect of eIF2 alpha O-GlcNAcylation on cell recovery, migration, accumulation of reactive oxygen species (ROS), and metabolic labeling during protein synthesis under different Arg conditions. Results Our research identified eIF2 alpha, eIF2 beta, and eIF2 gamma, as key O-GlcNAcylation targets in the absence of Arg. We found that O-GlcNAcylation of eIF2 alpha plays a crucial role in regulating antioxidant defense by suppressing the translation of the enzyme HO-1 during Arg limitation. Our study showed that O-GlcNAcylation of eIF2 alpha at specific sites suppresses HO-1 translation despite high levels of HMOX1 transcription. We also found that eliminating eIF2 alpha O-GlcNAcylation through site-specific mutagenesis improves cell recovery, migration, and reduces ROS accumulation by restoring HO-1 translation. However, the level of the metabolic stress effector ATF4 is not affected by eIF2 alpha O-GlcNAcylation under these conditions. Conclusions Overall, this study provides new insights into how ArgS fine-tunes the control of translation initiation and antioxidant defense through eIF2 alpha O-GlcNAcylation, which has potential biological and clinical implications.