Project Summary The NLR (nucleotide-binding domain, leucine-rich repeat containing protein, also known as NOD-like receptor) family is a diverse group of proteins that functions to regulate host immunity. Many of the NLRs have been shown to regulate inflammasome formation and control caspase-1 activation, IL-1β/IL-18 secretion and inflammatory cell death. In contrast a subset of NLR proteins negatively regulates different inflammatory pathways, typically via inhibitory interactions with key signaling molecules. Nonetheless the molecular mechanisms that control the negative regulation of inflammation are poorly understood. NLRC3 in particular has been shown to negatively regulate several key inflammatory pathways. For example, NLRC3 has been shown to inhibit NF-κB and PI3K pathways in response to TLR (or IGF-1R) signaling and proinflammatory cytokine secretion; alternatively, NLRC3 inhibits type I interferon production in response to cytosolic nucleic acid stimulation by regulating STING trafficking. The molecular mechanisms to allow for NLRC3 to function in diverse pathways are poorly understood. Understanding the molecular mechanisms of negative regulation of inflammation will be necessary for the generation of new strategies to improve health. Aim 1 of my proposal uses classic biochemical and cell biological techniques to examine the molecular determinants of novel protein-protein interactors with NLRC3. Our studies suggest that one of these interactors negatively regulates type I interferon production. In aim 1 of our proposal we will generate recombinant cell lines via CRISPR/Cas9, shRNA and overexpression to probe the specific role of each PPI. In aim 2 we will investigate the molecular mechanism of innate immune signaling pathways that are associated with NLRC3