Pellino E3 ubiquitin protein ligase 1 (PELI1) was identified as a binding partner of receptor-interacting protein kinase-3 (RIPK3) in proteome microarrays (Choi SW et al. 2018). Endogenous PELI1 was found to interact with RIPK3 and RIPK1 in the necrosome complex upon necroptotic stimuli treatment in both human colorectal adenocarcinoma cells (HT-29) and mouse embryonic fibroblasts (MEF). PELI1:RIPK3 interaction was further studied by iImmunoprecipitation combined with site-directed mutagenesis assays using necroptosis-resistant RIPK3-deficient human embryonic kidney 293T (HEK293T) and human cervical cancer-derived HeLa cells, which were transfected with the epitope-tagged RIPK3 and PELI1 proteins. PELI1 was found to bind RIPK3 through its forkhead-associated (FHA) domain. Phosphorylation of RIPK3 at T182 was necessary for PELI1 recruitment (Choi SW et al. 2018).

MEFs lacking PELI1 have increased RIPK3 and MLKL phosphorylation, and necroptosis in response to necroptotic stimuli. Lung tissues from PELI1 transgenic mice showed a decrease in basal MLKL phosphorylation indicating that upregulated PELI1 may function to preferentially remove activated RIPK3 and reduce MLKL phosphorylation in vivo. In addition, PELI1 reduced endogenous RIPK3 in human lung adenocarcinoma (H2009) and human B lymphoblastoid (Raji) cell lines; conversely, knockdown of PELI1 in HT-29 and RIPK3 (ectopic)-expressing HeLa cell lines led to increased RIPK3 protein without affecting RIPK3 mRNA expression. Proteasome inhibitors (MG132 and BTZ), but not by lysosome inhibitors, prevented PELI1-facilitated degradation of stably expressed RIPK3 in HeLa cells and endogenous RIPK3 in HT-29 cells. In keratinocytes from toxic epidermal necrolysis (TEN) patients, PELI1 expression is low and inversely correlated with RIP3 protein, suggesting that reduction in PELI1 leads to upregulated RIP3 expression, thus contributing to disease progression. Thus, PELI1 is thought to control RIPK3 protein destabilization via ubiquitylation-dependent proteasome-mediated degradation (Choi SW et al. 2018).