ATP-CITRATE LYASE expression and activity are modulated by O-GlcNAc levels

Abstract

The enzyme ATP-citrate lyase (ACLY) is a nucleocytoplasmic protein that cleaves citrate into oxaloacetate and acetyl-CoA. It is described as a central enzyme of cellular metabolism and has an important role in the stress response particularly in sepsis. Its activity depends on its phosphorylation (particularly that of phospho Ser447-Thr451-Ser455) and multimerization. We have identified ACLY among the cardiac O-GlcNAcylated proteins in young septic rats. Interestingly, ACLY was less O-GlcNAcylated when global O-GlcNAcylation levels were stimulated with NButGT treatment. The O-GlcNAcylation (O-GlcNac) is a post-translational modification (PTM) notably involved in cell survival, metabolism and stress response. This PTM is regulated by a unique enzyme couple : O-GlcNac Transferase (OGT) and O-GlcNacAse (OGA) and could compete with phosphorylation on proteins. O-GlcNAc stimulation improves outcomes in young septic rats. Yet, no studies have attempted to understand the potential link between O-GlcNAc and ACLY. Decipher the impact of the O-GlcNAcylation on the expression and activity of ACLY. Chinese hamster ovary cells (CHO) were treated for 24 h with 10 μM Thiamet G (OGA inhibitor) or 50 μM OSMI (OGT inhibitor). O-GlcNAc levels, ACLY, P-ACLY Ser 455 et P-ACLY T447-S451 protein expression were evaluated by Western blot. ACLY activity was measured using the malate dehydrogenase coupled method. All results were compared to untreated CHO. Treatment with Thiamet G increases protein O-GlcNAcylation levels by 36% while treatment with OSMI decreases protein O-GlcNAcylation by 2 folds. Thiamet G does not affect ACLY's expression, phosphorylation or activity, while OSMI increases the expression of ACLY by 44% and in particular its multimers by 3 folds and all site of phosphorylation (P447-451-455). Accordingly, ACLY activity is also increased (+19%). In CHO cells, increasing the levels of O-GlcNAcylation of proteins with an OGA inhibitor has no impact on ACLY expression and activity, whereas decreasing the levels of O-GlcNac results in a significant increase in the expression of ACLY, its phosphorylation, multimerization and activity. We report for the first time the regulation of ACLY via the O-GlcNAcylation.