The inflammasome is a macromolecular complex that activates Caspase-1, a cysteine proteinase responsible for processing several cytokines (IL-1? and IL-18) into active, mature forms that are released from cells. In addition to cytokine processing, the inflammasome induces a novel cell death program with morphologic and biochemical features of necrosis. The ATP-binding protein NLRP3 acts as a central scaffold during in the assembly of some inflammasomes, termed NLRP3-inflammasomes. The NLRP3-inflammasome can be activated by numerous Pathogen-Associated and host derived Damage-Associated Molecular Patterns (PAMPs and DAMPs) as well as mutations in the ATP-binding domain that are associated with hereditary periodic fever syndromes. The common molecular mechanisms that control NLRP3 ATP-binding and inflammasome assembly in response to a wide variety of toxins have remained elusive. We have recently demonstrated that NLRP3 binds directly to oxidized DNA, a molecule that has been implicated in activation of NLRP3 by some stimuli. Preliminary data suggests binding accelerates the ATPase activity of NLRP3. We now propose to study the molecular mechanisms of NLRP3 activation during physiologic and pathologic activation states. We propose: 1) to determine the role oxidized DNA binding in the ATP binding cycle and inflammasome assembly using in vitro reconstitution of the NLRP3-inflammasome and to define the DNA binding domain of NLRP3. 2) to explore whether NLRP3 is activated by other oxidized polynucleotides including RNA and DNA/RNA hybrids (which have also been implicated in NLRP3 activation) and identify the polynucleotides that associate with NLRP3 when it is activated in cells by different physiologic and pathologic stimuli. 3) to determine the role of NLRP3 phosphorylation in controlling oxidized DNA binding, ATPase activity, and inflammasome assembly.
Statement: The NLRP3-inflammasome is signaling complex that is activated in a number of inflammatory and infectious conditions, including severe necrotizing infections caused by antibiotic resistant Staphylococcus aureus. Activation of the NLRP3 inflammasome in these diseases is often pathologic, driving tissue damage and disease progression. We propose to determine the molecular mechanisms that drive activation of this signaling complex which will help to define potential targets for new therapies to treat these diseases.
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