PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP

Abstract

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.

Post-translational Modification of OTULIN Regulates Ubiquitin Dynamics and Cell Death

“…Blocking OTULIN Tyr-56 phosphorylation did not affect the assembly of the necrosome ( Figure 5D), which is consistent with previous reports demonstrating that LUBAC deficiency affected RIPK1 ubiquitination without changes to complex formation kinetics (de Almagro et al, 2015). Necroptotic RIPK1 ubiquitination by the E3 ligase Pellino-1 was found to be dependent on RIPK1 auto-phosphorylation (Wang et al, 2017a). Consistently, a kinase-dead RIPK1 mutant was deficient in Met1-Ub and Lys63-Ub (de Almagro et al, 2017), suggesting that RIPK1 phosphorylation precedes its ubiquitination.…”

Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target

“…Deficiency of PELI1 disrupts the interaction between RIPK1 and RIPK3 and abrogates necroptosis, suggesting that PELI1 might function as a mediator in this process. PELI1-induced Lys63 ubiquitination of RIPK1 on Lys115 was confirmed, which was dependent of the RIPK1 kinase activity [34]. Taken together, this implies that phosphorylation, dimerization and ubiquitination of RIPK1 are essential for inducing necroptosis.…”

“…Finally, ubiquitination of RIPK1 is essential for its regulation and the interaction with RIPK3. Besides the polyubiquitination by cIAPs and LUBAC to form complex I, and the deubiquitination by CYLD and A20 to form complex II, pellino E3 Ubiquitin Protein Ligase 1 (PELI1) also regulates the function of RIPK1 [34]. Deficiency of PELI1 disrupts the interaction between RIPK1 and RIPK3 and abrogates necroptosis, suggesting that PELI1 might function as a mediator in this process.…”

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

“…Other innate immune kinases, such as IRAK1 (Interleukin‐1 (IL‐1) receptor‐associated kinase 1) and IRAK4, are ubiquitination targets for other Ub ligases such as TRAF6 and Pellino1 in TLR/IL‐1R signaling (Cohen & Strickson, ). Remarkably, knowledge about the substrates of RIPK and IRAK kinases is limited and there is a continuing debate about the role of these proteins as true kinases versus their scaffolding function, including as scaffolds for ubiquitination (Kawagoe et al , ; Kim et al , ; Koziczak‐Holbro et al , ; Ordureau et al , ; Song et al , ; Vollmer et al , ; Wang et al , ; Annibaldi & Meier, ; Dziedzic et al , ). Our findings for RIPK2 exemplify the cross‐talk between the kinase domain serving as a binding interface for a Ub ligase (XIAP) and kinase inhibitors acting as blockers of this interaction.…”