Project Summary/Abstract NACHT, LRR and PYD domains-containing protein 3 (NLRP3) belongs to a class of Nod-like-receptor (NLR) proteins that trigger the assembly of the inflammasome, a molecular platform that mediates caspase-1 activation and processing and secretion of biologically active IL-1? and IL-18. In addition to its critical role in innate immunity, dysregulation of the NLRP3 inflammasome has been linked to both inherited and acquired inflammatory disorders, such as Cryopyrin-associated autoinflammatory syndrome, gout, Crohn?s disease, Alzheimer?s disease, diabetes and atherosclerosis. Despite the medical importance of the NLRP3 inflammasome, the mechanism by which it is activated remains elusive. Using a proteomics approach to reveal critical factors that interact with NLRP3 in macrophages, we identified the Nek7 protein kinase as a NLRP3- interacting protein that is essential for the assembly of the NLRP3 inflammasome. Nek7 is associated with NLRP3 in the resting state, and this interaction is enhanced during NLRP3 activation. In macrophages depleted with Nek7, caspase-1 activation and IL-1? secretion are abrogated in response to stimuli that trigger NLRP3 activation. In contrast, Nek7 is not required for the activation of the NLRC4 and AIM2 inflammasomes. The critical and specific role of Nek7 in the activation of the NLRP3 inflammasome is unexpected because Nek7 is a member of a kinase family that regulates cytokinesis. The goal of this proposal is to define the molecular mechanism by which Nek7 regulates the activation of the NLRP3 inflammasome. Based on our preliminary results, our primary hypothesis is that the Nek7 protein kinase regulates NLRP3 inflammasome activation by promoting the assembly of the inflammasome. Furthermore, we hypothesize that Nek7 contributes to the induction and/or progression of inflammatory disease. To test these hypotheses, we propose characterizing the mechanism by which Nek7 regulates NLRP3 inflammasome activation and the role of Nek7 in vivo using animals models of inflammation that rely on IL-1? secretion via NLRP3. Understanding the role of Nek7 in NLRP3 inflammasome activation is expected to provide critical insight into the development of novel therapeutic strategies for inflammatory diseases.