Apoptosis Induced by the Toll-Like Receptor Adaptor TRIF Is Dependent on Its Receptor Interacting Protein Homotypic Interaction Motif

Abstract

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-β (TRIF), TRIF-related adapter molecule (TRAM), and sterile α motifs and β-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-κB activation. The domain that was required for induction of apoptosis could activate NF-κB but not IFN regulatory factor-3, yet the activation of NF-κB could be blocked by superrepressor IκBα without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-κB but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.

RIP1, a kinase on the crossroads of a cell's decision to live or die

“…52 The RHIM domain in the C-terminus of TRIF is required for the induction of apoptosis. 53 TRIF-induced apoptosis was blocked by dominant-negative FADD and caspase-8 and by the caspase inhibitors zVAD-fmk and CrmA, indicating that TRIF induces an apoptosis pathway that is dependent on RIP1/FADD/ caspase-8. TRIF-dependent apoptosis has been confirmed for TLR4 and TLR3 agonists.…”

RIP kinases: key decision makers in cell death and innate immunity

“…109 In addition, some of these pathways have been associated with cell apoptosis. Overexpression of TRIF results in interaction with RIP1 and RIP3 and induction of apoptosis, 110 and in the context of TLR3 signalling in lung cancer cells, the TLR3 ligand dsRNA can induce apoptosis by recruiting caspase 8 to TLR3 in a RIP1-dependent manner. 111 The ability of RIP kinases to orchestrate both apoptosis and necroptosis in response to triggering of viral-sensing TLR3 provides a major survival advantage to the host.…”

The caspase-generated fragments of PKR cooperate to activate full-length PKR and inhibit translation

“…This suggests that the cell death pathways studied here were initiated by the interaction of dsRNA with a cell surface receptor such as TLR3. 20 Although our current results suggest that the catalytic activity of PKR does not play a major role in dsRNA-induced necrosis, we cannot exclude that the protein is required in this cell death process. In addition to eIF2-a phosphorylation and inhibition of translation, PKR was reported to control the activation of several transcription factors such as NF-kB, p53, and STATs.…”

“…19,20 We demonstrated here that RIP1 is probably required not only for dsRNA-induced signaling to apoptosis but also for dsRNAinduced necrosis.…”

“…In addition to NF-kB and IRF-3 activation, 19 signaling to apoptosis in response to external dsRNA was suggested to be TLR3 TIRadapter (TRIF)-dependent and to occur via interaction with the RIP1 and Fas/Apo-1-associated death domain (FADD), thus leading to caspase-8 activation. 20 When we transiently coexpressed green fluorescent protein (GFP) with either TRIF, RIP1, FADD, or caspase-8 in HEK293T cells, transfected cells died by apoptosis (data not shown).…”