Scription factor three (IRF3), indicating that NLRC3 most likely functions in the upstream
Scription element 3 (IRF3), indicating that NLRC3 probably functions in the upstream STING-TBK level (Figure 3A). As a specificity control, a further NLR, NLRP11, didn’t decrease IFN- promoter activation by TBK1 (Figure 3B). NLRC3 also inhibited a second promoter driven by the canonical interferon-stimulated responsive element (ISRE), which is identified to be activated by STING and TBK1 (Ishikawa and Barber, 2008; Zhong et al., 2008) (Figure 3C). Even so NLRC3 had no impact on the activation from the ISRE promoter by mitochondrial antiviral signaling protein (MAVS) (also referred to as interferon-beta promoter stimulator 1 (IPS-1), virus-induced signaling adapter (VISA) and CARD adaptor inducing IFN- (CARDIF)), which is crucial for RNA sensing, nor did it affect promoter activation by the downstream IRF3 (Figure 3C). Furthermore, NLRC3 inhibited NF-B promoter activated by STING, and lowered MAVS activation slightly but did not influence retinoic acid-inducible gene 1 (RIG-I)(Figure 3D). We also observed that NLRC3 inhibited c-di-GMP and poly(dA:dT)-induced ISRE activation (Figure 3E). These experiments indicate that the predominant effect of NLRC3 is around the STING pathway. As an extra specificity handle for NLR proteins, MDM2 Inhibitor supplier overexpression of NLRC5, which has been reported to inhibit different innate immune pathways when tested in an overexpression system (Cui et al., 2010) did not inhibit STING or TBK1-induced ISRE activation (Figure 3F). These experiments suggest that NLRC3 down-regulates innate immunity caused by STING and TBK1.Immunity. Author manuscript; offered in PMC 2015 March 20.Zhang et al.PageNLRC3 associates with STING and TBK1 and alters the STING-TBK1 interaction just after stimulationNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptTo explore the mechanism by which NLRC3 interferes with STING and TBK1 function, we tested if NLRC3 interacts with STING andor TBK1. Transient transfection and co-immunoprecipitation followed by immunoblot showed that HA-NLRC3 strongly related with Flag-STING and more modestly with TLR7 Agonist Compound Flag-TBK1, but not with Flag-IRF3 (Figure 4A), suggesting it interacts with all the upstream STING-TBK complicated but not together with the downstream IRF3. This agrees with earlier data indicating that NLRC3 affected STING and TBK1 function but not IRF3 function (Figure 3A). Immunoblot of the input protein indicates that all of the proteins are expressed in readily detectable amounts (Figure 4A, appropriate panel). Within a far more physiologic approach, HA-NLRC3 also associated with endogenous STING (Figure 4B, top lane) and TBK1 (Figure 4C) in a hemi-endogenous system, but not with IRF3 (information not shown). These experiments indicate that NLRC3 can associate with STING and TBK1. To additional investigate whether the association among NLRC3 and STING is direct, we ready purified, recombinant complete length NLRC3 and truncated STING protein (amino acid 13979 and 13944) and performed a protein pull-down assay. The results show NLRC3 and STING directly bind to each other within a reciprocal pull-down assay (Figure 4D ). Next, a domain mapping experiment was carried out with NLRC3 deletion constructs (Figure 4F). Full-length NLRC3, caspase activation and recruitment domain (CARD)nucleotide binding domain (NBD) and NBD strongly connected with STING, even though the CARD or leucine-rich repeats (LRR) domain alone either did not associate, or didn’t associate strongly, with STING (Figure 4F). The CARD domain alone did not express in high amounts, having said that.
