Background: RIP3-dependent programmed necrosis is an alternative to apoptosis. Results: When caspase-8 is compromised, TRIF-dependent TLRs directly activate RIP3 kinase through RHIM-dependent interactions. Conclusion: TRIF mediates direct RHIM-dependent signaling, triggering necrosis via RIP3 and MLKL. Significance: Programmed necrosis eliminates cells following stimulation of either MyD88 or TRIF signaling pathways that converge on RIP3.

Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

Limited proteolysis of Gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or −11. It is currently unknown if macrophage GSDMD can be processed by other mechanisms. Here we describe an additional pathway controlling GSDMD processing. The inhibition of TGF-β activated kinase-1 (TAK1) or IKK kinases by the Yersinia effector protein YopJ elicits RIPK1-and caspase-8-dependent cleavage of GSDMD which subsequently results in cell death. GSDMD processing also contributes to the NLRP3 inflammasome-dependent release of IL-1β. Thus, caspase-8 acts as a regulator of GSDMD-driven cell death, and our study establishes the importance of TAK1 and IKK activity in the control of GSDMD cleavage and cytotoxicity.

Summary Receptor interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pro-necrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound; whereas, D161G, D143N, and K51A mutants only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/D161N mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

No abstract