A New Domain in the Toll/IL-1R Domain-Containing Adaptor Inducing Interferon-ß Factor Protein Amino Terminus Is Important for Tumor Necrosis Factor-a Receptor-Associated Factor 3 Association, Protein Stabilization and Interferon Signaling

Toll/IL-1R domain-containing adaptor inducing interferon-β (IFN-β) factor (TRIF) is a key adaptor for Toll-like receptor (TLR) 3 and TLR4 signaling. Using a novel cDNA isolate encoding a TRIF protein with a 21-residue deletion (Δ160-181) from its amino-terminal half, we investigated the impact of this deletion on TRIF functions. Transfection studies consistently showed higher expression levels of the (Δ160-181) TRIF compared to wild-type (wt) TRIF, an effect unrelated to apoptosis, cell lines or plasmid amplification. Colocalization of wt and (Δ160-181) TRIF proteins led to a dramatic reduction of their respective expressions, suggesting that wt/(Δ160-181) TRIF heterocomplexes are targeted for degradation. We demonstrated that wt TRIF associates with tumor necrosis factor-α receptor-associated factor 3 (TRAF3) better than (Δ160-181) TRIF, culminating in its greater ubiquitination and proteolysis. This explains, in part, the differential expression levels of the two TRIF proteins. Despite higher expression levels in transfected cells, (Δ160-181) TRIF inefficiently transactivated the IFN pathway, whereas the nuclear factor-κB (NF-κB) pathway activation remained similar to that by wt TRIF. In coexpression studies, (Δ160-181) TRIF marginally contributed to the IFN pathway activation, but still enhanced NF-κB signaling with wt TRIF. Therefore, this 21 amino acid sequence is crucial for TRAF3 association, modulation of TRIF stability and activation of the IFN pathway.