NLRs represent the intracellular analog to the TLRs (Toll-like receptors), important mediators of innate immunity. Oligomerized NLRs serve as platforms for activating signaling proteins, including inflammatory Caspases involved in cytokine processing and apoptosis. These proteins complexes have been dubbed, "inflammasomes." This renewal application focuses on NLR-family member NLRP1 (NALP1) as a prototype of the NLR family. During the previous funding period, we reported that the leucine-rich repeats (LRRs) of NLRP1 are required for responses to microbial ligands derived from peptidoglycan, analogous to the LRRs of TLRs in sensing pathogens. We also demonstrated that the LRRs inhibit the NACHT domain from binding nucleotide triphosphates (NTPs) and from oligomerizing, until stimulated by microbial ligands. We also learned that activation of NLRP1 is inhibited by Bcl-2-family members, proteins involved in apoptosis regulation. Recently, we obtained evidence that viral homologs of Bcl-2 encoded in poxviruses bind NLRs, suggesting a mechanism by which they suppress host inflammatory responses. Also, polymorphisms in NLRP1 in mice have been causally linked to differential sensitivity to bacterial virulence factors, including anthrax toxin (Lethal Factor [LF]), a zinc metalloproteinase that depends on NLRP1 for inducing Caspase-dependent killing of macrophages, thereby impairing host defense. We have found that LF directly cleaves and activates NLRP1 in vitro. Moreover, hereditary polymorphisms in human NLRP1 are associated with vitiligo with autoimmune features. Based on these observations, we have proposed the hypothesis that NLRs are central integrators of inflammatory responses to pathogens and cell stress. Our goals for this competitive renewal application are to address the following questions about NLR-family proteins, focusing on NLRP1 as a prototype: (1) What is the mechanism by which bacterial ligands activate the NALP1 inflammasome and how is this mechanism different for the hereditary mutants of NLRP1 associated with autoimmunity?;(2) What is the mechanism by which anthrax toxin [LF] activates NLRP1 and how does this compare with bacterial ligands?;(3) What is the mechanism by which Bcl-2 and Bcl-XL suppress NLRP1 activation induced by bacterial peptidoglycan products and what are the implications for learning how to regulate NLR proteins?;and (4) What are the effects of viral Bcl-2 homologs on NLRP1 and how does this impact host defense against viruses? Answers to these questions will provide insights into the mechanisms regulating NLR-family proteins, laying a foundation for addressing unmet medical needs in the areas of autoimmunity, allergy, inflammation, vaccines, and infectious diseases, while also aiding in development of counter measures for bioterrorism. PUBLIC HEALTH RELEVANCE NLR proteins constitute a large group of proteins involved in host-defense, protecting our body from bacteria and viruses as a first-line of defense, known as innate immunity. Imbalances in the endogenous mechanisms that regulate NLR-family proteins are associated with inflammatory and autoimmune diseases, where these proteins become hyperactive. Conversely, susceptibility to infection is associated with insufficient or inappropriate activity of these proteins. NLRs are attacked by proteins encoded in the genomes of bacteria and viruses, thereby thwarting immune responses. The bacterial and viral pathogens known to attack NLRs include agents of bioterrorism, including Anthrax Toxin and poxviruses. This proposal seeks to improve understanding of the mechanisms regulating NLRs, including the endogenous cellular mechanisms that keep them in check, and the mechanisms relevant to bacterial and viral infection. The proposed project is relevant to several diseases with unmet medical needs, including autoimmunity, chronic inflammation, allergy, infection, and vaccines. Moreover, the recent discovery that NLRs are activated by tissue injury suggests their involvement also in pathological inflammatory responses in the context of trauma, burns, and ischemic diseases such as stroke and heart attack. Thus, studies of NLRs have relevance to a wide diversity of human diseases.