MECHANISMS OF ACTIVATION OF ICE-LIKE CYSTEINE PROTEASES

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DESCRIPTION (provided by applicant): Caspase-1 is an important component of the innate immune response against pathogenic infection and cellular stresses. Activation of caspase-1 can lead to processing of the inactive pro-IL-12 and pro-IL-18 to produce the active cytokines IL-12 and IL-18, respectively, which are potent mediators of inflammation that stimulate fever, recruitment and activation of immune cells, production of secondary cytokines and cellular proliferation. Caspase-1 activation can also lead to an inflammatory form of cell death called pyroptosis. The applicant's laboratory demonstrated recently that this form of cell death is mediated by the caspase-1 adaptor protein ASC, which assembles a supramolecular structure termed the pyroptosome that activates caspase-1 during pyroptosis in response to diverse pro-inflammatory and stress signals. They have also elucidated the role of pyrin, a protein mutated in familial Mediterranean fever, in caspase-1 activation and inflammation in the human auto-inflammatory PAPA syndrome, and demonstrated that pyrin is a homotrimeric cytosolic receptor for the cytoskeleton- organizing protein PSTPIP1. Ligation by PSTPIP1 activates pyrin by unmasking of its PYD domain, which then interacts with ASC and facilitates ASC oligomerization into the active pyroptosome. In this application specific aims are proposed to extend these ongoing studies to further investigate the regulation and molecular determinants of the interaction between PSTPIP1 and pyrin. In particular, the aims will focus on characterizing the exact role of the protein-tyrosine phosphatase PTP-PEST and tyrosine kinase c-Abl, both of which have been shown to interact with PSTPIP1, in the pyrin-mediated caspase-1 activation pathway. It is also proposed to characterize novel upstream activators of the ASC pyroptosome and further investigate the role TLR2, TLR4, cryopyrin and caspase-1-mediated proteolysis in macrophage pyroptosis. Finally, studies are proposed to investigate the potential use of the in vitro ASC pyroptosome assembly as a high throughput screen to identify small molecule therapeutics to inhibit and treat inflammation. PUBLIC HEALTH RELEVANCE: Inflammation is a key process in the innate immune response to infections and cellular stress. This research focuses on understanding the role of key cellular proteins in the complex molecular and cellular inflammatory mechanisms. The results of this research will help in the design and discovery of effective therapeutics to treat inflammation associated with aging, auto-inflammatory disease and chronic inflammatory diseases.