The innate immune system protects the host from infections, detects and repairs tissue damage and functions to maintain tissue homeostasis. Several families of signaling receptors can recognize microbial substances or altered host molecules and orchestrate a coordinated inflammatory response. Inflammasomes are signaling platforms that control proteolytic activation of highly proinflammatory cytokines of the IL-1β family and thus, are relevant for infection control and numerous inflammatory conditions. In addition to recognizing foreign signals, the NLRP3 inflammasome can sense sterile tissue damage and various endogenous danger signals that appear in many common chronic inflammatory conditions. NLRP3 can be triggered by material released from dying cells and aggregated or crystalline substances, and its activation has been implicated in the pathogenesis of prevalent diseases in Western societies, such as type 2 diabetes, COPD, atherosclerosis and Alzheimer’s disease. The NLRP3 inflammasome can be activated by diverse signals however, the molecular mechanisms leading to its activation remain poorly understood. Using chemical biology screens and proteomics analysis, we identified that NLRP3 activity is regulated by phosphorylation and ubiquitination. This project aims to identify the enzymes and signaling mechanisms leading to NLRP3 activation. In an integrated, multidisciplinary approach, we will employ chemical biology screening to identify novel targets that act to regulate NLRP3, and will describe the NLRP3 interactome in response to various triggers. Data obtained by these approaches will be analyzed by bioinformatics, and signaling mechanisms identified will be confirmed by RNA interference and gain-of-function studies. Utilizing a range of biochemical, biophysical and immunological techniques, we will determine the mechanisms by which the identified molecules can activate the NLRP3 inflammasome and assess their physiological relevance in models of inflammation.