The Interaction Of IFN-λR1 With TRAF6 And The Effect Of NF-κB And ISRE Activity

Interferon-lambdas (IFN-λs) is a newly identified IFN family whose genome structure is familiar to IL-10members and its charactors is related to the type Ⅰ IFNs. IFN-λS act through a cell surface receptor composed of two chains, the first one being IFN-λ specific and the second one shared with IL-10, which belongs to the type Ⅱ cells factor receptor family. IFN-λS lead a cell signal through IFN-λR1and activate the Jak-STAT pathway as the type Ⅰ IFN. When the IFN-λ ligand binding to IFN-λR1, the Jak-STAT pathway can be activited and then the STAT1, STAT2, STAT3, STAT4and STAT5are phosphorylated. Then, the phosphorylated STAT1and STAT2bind to p48formed a trimer complex:ISGF3. ISGF3bind to the cis-element ISRE and start to express genes like OAS1, Mx1to antiviral. Dual Luciferase Reporter Gene Assay was used to research ISRE activities.This charactor made IFN-Xs show a similar to type I IFN: antivirus, anti-proliferative activity and immune regulation. Unlike the type Ⅰ IFN receptor, the expression of IFN-λR1is tissue specific, there is low expression in the bone marrow and brain spinal cord. The anti-proliferative activity of IFN-λs is slightly weaker than type Ⅰ IFN, which makes the IFN-λS less side effection in central nervous system and bone marrow. These features make IFN-λs a giant and potential clinical application.We found that the IFN-λR1has a consensus motif which can interact with TRAF6through the amino acid analysis. And we proved this interaction in our preliminary works. In order to further explore what kind of biological effect will happen under the interaction between IFN-λR1and TRAF6, we first produced the recombinant human protein IFN-λ1(rhIFN-λ1) in our laboratory by using Pichia pastoris Yeast system. The expressed rhIFN-λ1was purified by cation exchange and gel filtration chromatography. Then, the expression vector containing the full length IFN-λR1cDNA with signal peptide coding sequences was build which can express IFN-λR1with a tag of Myc, or GFP and Flag. Immunofluorescence and membrane protein sepration experiments were employed to comfirm the expression of IFN-λR1. Co-IP showed that IFN-λR1can interact with TRAF6for a long time under the stimulation of rhIFN-λ1and in15min the interaction was decreased. When IFN-λR1was co-transfect with TRAF6, the activity of NF-κB was repressed and the p38was dephosphorylated. TRAF6can pass the ubiquitin from UBC13/UEV1A to the target protein through a RING motif in its N-terminal domain. The IFN-λR1was ligased with Lys-63ubiquitin by TRAF6and rhIFN-λ1could promote this action. And the OAS1and Mx1mRNA levels were detected by real-time quantitative PCR. Both of this two experiments showed that TRAF6can inhibit ISRE activity and down-regulate the OAS1, Mxl mRNA expression level.This part of the experiment provided some evidences to indicate that TRAF6is a new binding protein of IFN-λR1.