Cloning and characterization of novel nod like receptors as cytoplasmic immune sensors

Abstract

NOD like receptors recognize infection and cellular damage. They are characterized by an N terminal protein interaction domain, central oligomerization domain, termed nucleotide binding domain and leucine rich repeat domain at the C-terminus. NLRC3 protein has the characteristic NBD-LRR configuration with a CARD domain. T lymphocytes have the highest expression level of NLRC3. It was suggested that it functions as a novel suppressor of T cell activation. NLRP13 shares the NBD-LRR configuration, but contains a PYRIN domain. Placenta is the only known tissue to express NLRP13. We found that both EGFP-NLRC3 and EGFP-NLRP13 fusion proteins are mainly localized in the cytoplasm but they can also be localized in mitochondria; therefore, they can be considered as cytoplasmic immune sensors. To determine the possible interactions of NLRC3 and NLRP13 with inflammasome components, coimmunoprecipitation experiments were performed. Co-IP results suggest that both NLRC3 and NLRP13 can interact with ASC and Caspase 1. The outcomes of Co-IP were verified with co-localization of ASC and Caspase 1 with NLRC3 and NLRP13 in HEK293FT cells via confocal microscopy. Under confocal microscopy, we detected for the first time that inflammasome like complexes assembled when Caspase 1 and ASC were co-transfected with NLRC3 or NLRP13 in HEK293FT cells. One of the Co-IP results suggests that NLRC3 can also interact with Caspase 5 and RFP NLRC3 co-localized with EGFP Caspase 5 in the HEK293FT cells. Dose and time dependent transfection of NLRC3 induces the speck formations of ASC in the stable EGFP ASC HEK293FT cells. However, NLRP13 does not cause speck formation as efficiently as NLRC3. To test the effects of NLRC3 and NLRP13 on NF-κB signaling, luciferase reporter gene assay has been performed. Overexpression of NLRC3 and NLRP13 proteins resulted in NF-κB downregulation.