Malonylation of GAPDH in the inflammatory response in macrophages

Abstract

Succinylation and malonylation are two recently discovered protein post-translational modifications. They involve the attachment to lysines of metabolism-derived succinyl and malonyl groups respectively, resulting in a change in charge comparable to that of phosphorylation. Given the increasing number of links existing between the metabolic status and the effector function of immune cells, I investigated whether protein succinylation and malonylation might play a role in the activation of macrophages during metabolic reprogramming. Protein succinylation and malonylation were observed to be induced in murine macrophages following activation with various Toll-Like Receptor (TLR) ligands. I was able to identify via mass spectrometry a wide array of proteins undergoing these post-translational modifications, one of which was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lipopolysaccharide (LPS) induced malonylation of GAPDH lysine 213, within the catalytic domain of 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 as well as siRNA knockdown, I found that GAPDH activity was needed for the production of cytokines in macrophages, including TNF?, IL6 and IL1?. However, especially in the case of TNF?, the requirement for GAPDH went beyond its glycolytic activity. Following activation of macrophages with LPS, GAPDH dissociated from TNF? mRNA, enabling its translation. Furthermore, GAPDH enzymatic activity and RNA binding were mutually exclusive, as revealed using heptelidic acid (HA), an inhibitor of enzymatic activity. HA increased the repressory binding of GAPDH to mRNAs, inhibiting TNF? production. A mutant form of GAPDH (GAPDH K213E), which mimicked malonylation was more active enzymatically but less able to bind the mRNA for TNF?. A form that is unable to undergo malonylation, GAPDH K213Q, bound more tightly to the mRNA, but was less enzymatically active. Overall, these results show that malonylation increased GAPDH enzymatic activity, which is needed for the production of cytokines such as IL1? and IL10. At the same time, mRNAs bound to GAPDH, including that of TNF?, are released and translated. Therefore, malonylation of GAPDH is an important signal for macrophage activation.