Transglutaminase type 2 regulates necroptosis

Necroptosis is a mechanism of programmed cell death that shows features in common with both apoptosis and necrosis. From the molecular point of view necroptosis can be induced by different molecules such as Tumour Necrosis Factor a (TNFa), Fas ligand, interferon a, b, g and others. The binding of the TNFa to its receptor results in the formation of the complex IIb. In this complex, receptor-interacting serine/threonine-protein kinase 1 (RIPK1) promotes the phosphorylation of RIPK3. RIPK3 can in turn phosphorylate MLKL that oligomerizes through disulphide bonds moving from the cytosol to the plasma membrane forming pores that produce the rupture of the same and leading to cell death. Here, we demonstrated that Transglutaminase type 2 protein (TG2) can regulate necroptosis pathway favouring the expression of Ripk3 gene. TG2 is a multifunctional enzyme that moves from a closed to an open form in response to elevated intracellular Ca2+ concentrations, leading to the activation of its cross-linking and transamidase activities. TG2 has also calcium independent activity such as G-protein, scaffold, kinase, and protein disulphide isomerase activity. TG2 through its multiple activities can regulate different transcriptional factors and it can also post-translationally modify the H3 histone (H3K4me3Q5ser) by serotonylation thus promoting gene expression. Starting from an RNA seq analysis performed in mouse embryonic fibroblasts (MEF) derived from wild type mice (WT MEF) and TG2 knockout mice (TG2 KO MEF), we identified a strong downregulation of Ripk3 gene in absence of TG2. Interestingly, we observed that in cells lacking TG2, after necroptosis induction, RIPK3 and MLKL were not phosphorylated indicating that there was a defect in the activation of necroptosis. Moreover, TG2 KO MEF did not die after necroptotic stimulus compared to WT MEF that showed a reduced cell viability. Interestingly, we found that the absence of RIPK3 expression, in cells lacking TG2, was due to DNA methylation. Indeed, using 5-Aza-2'-Deoxicytidine that prevents DNA methylation, we restored Ripk3 gene expression in TG2 KO MEF. Moreover, we also observed that TG2 interacted with both histones H3K4me3 and H3K4me3Q5ser and more specifically, we found that these histone modifications were associated with the Ripk3 promoter in presence of TG2. All together these results indicate that TG2 is essential to promote the serotonylation on H3K4me3 allowing Ripk3 gene expression making cells susceptible to necroptosis.