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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI088255-10
Application #
9934970
Study Section
Immunity and Host Defense (IHD)
Program Officer
Davidson, Wendy F
Project Start
2010-05-01
Project End
2021-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Duncan, Joseph A; Canna, Scott W (2018) The NLRC4 Inflammasome. Immunol Rev 281:115-123
Melehani, Jason H; Duncan, Joseph A (2016) Inflammasome Activation Can Mediate Tissue-Specific Pathogenesis or Protection in Staphylococcus aureus Infection. Curr Top Microbiol Immunol 397:257-82
Ezekwe Jr, Ejiofor A D; Weng, Chengyu; Duncan, Joseph A (2016) ADAM10 Cell Surface Expression but Not Activity Is Critical for Staphylococcus aureus ?-Hemolysin-Mediated Activation of the NLRP3 Inflammasome in Human Monocytes. Toxins (Basel) 8:95
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Mo, Jinyao; Duncan, Joseph A (2013) Assessing ATP binding and hydrolysis by NLR proteins. Methods Mol Biol 1040:153-68
Taxman, Debra J; Swanson, Karen V; Broglie, Peter M et al. (2012) Porphyromonas gingivalis mediates inflammasome repression in polymicrobial cultures through a novel mechanism involving reduced endocytosis. J Biol Chem 287:32791-9

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