Malonylation is a recently uncovered protein post-translational modification. It involves the attachment of a malonyl-CoA-derived malonyl group to lysines, resulting in an extra 86 Da mass, as well as a change in charge comparable to that of phosphorylation. Given the increasing number of links existing between the metabolic status and the function of immune cells, it led us to investigate whether protein malonylation might be a new mechanism by which the cell’s metabolism can control the immune response.
We have been able to show that protein malonylation can be induced in macrophages following activation. We have also been able to identify via mass spectrometry a wide array of proteins undergoing this post-translational modification, one of which is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Although it is most widely known for its role as a glycolytic enzyme, GAPDH has blossomed in recent years from its mundane use as an endogenous control into an increasingly interesting multi-functional protein. Through the use of a GAPDH enzymatic activity inhibitor, and siRNA knockdown, we have shown that GAPDH activity is needed for the production of cytokines in macrophages, including TNFα, IL6 and IL1β. However, especially in the case of TNFα, the requirement for GAPDH goes beyond its glycolytic activity. GAPDH can bind TNFα mRNA, as well as other pro-inflammatory mRNAs, and block their translation. Following activation of macrophages with lipopolysaccharide (LPS), GAPDH can release these mRNAs and enable their translation.
Our data would suggest that there is a specific lysine within GAPDH catalytic domain that undergoes LPS-induced malonylation. By generating a malonylation-mimic mutant of this lysine, GAPDH activity is boosted while its binding to TNFα mRNA is inhibited. Overall, our results suggest that malonylation has important signalling functions in macrophages and can act as a key regulator of cytokine production and inflammation via GAPDH.