In vertebrates two subfamilies of caspases, the caspase-3-like and caspase-1-like caspases, play important roles in cell death and in inflammation, respectively. Unlike the significant progress which has been made in recent years towards understanding the mechanism of activation and regulation of the apoptotic caspase-3-like caspases, very little is known in this regard with respect to the pro-inflammatory caspase-1-like subfamily of caspases which include caspase-1, -4 and -5 in human. Caspase-1 is the major intracellular protease that cleaves the precursors of IL-1 beta and IL-18 into active cytokines. Recent observations suggest that procaspase-1 is activated through direct binding to the newly discovered adaptor protein Ipaf/CARD12. In addition other preliminary results suggest that the PYRIN-CARD protein ASC interacts with caspase-1 via CARD-CARD interactions. This observation implicates ASC as a central adaptor for activation of caspase-1 by the PYRIN-containing proteins DEFCAP/NAC/CARD-7 and cryopyrin/Pypaf1 which also interact with ASC via PYRIN-PYRIN domain interactions. Thus we propose that during inflammation procaspase-1 is recruited by Ipaf and/or ASC/DEFCAP/cryopyrin to a large """"""""inflammasome"""""""" complex(s) similar to the Apaf- 1-caspase-9 apoptosome in response to proinflammatory cytokines. To test this hypothesis experiments are proposed to study the mechanism of activation and regulation of caspase-1-like caspases by Ipaf and the other caspase-1-interacting PYRIN containing proteins. In particular, the first specific aim describes experiments to define the role of Ipaf in the mechanism of activation of caspase-1. These experiments employ RNA interference (RNAi) techniques to silence Ipaf expression in THP-1 cells, dominant negative Ipaf-mutant to interfere with caspase-1 function and finally structural analysis of the caspase-1 and Ipaf-caspase-1 complex. The second specific aim addresses the potential pro-inflammatory function and role of the PYRIN-domain containing proteins ASC, NAC and cryopyrin in the mechanism of caspase-1, -4 and -5 activation, through detailed structure-function analyses, RNAi experiments and characterization of protein-protein interactions. The third specific aim will address in more detail the mechanism of activation of caspase-1 by in vitro reconstitution of a caspase-1 activation system and identification of upstream regulators of the caspase-1 inflammasome. Combined these studies are expected to yield important insight into the mechanisms by which caspase-1 is activated and regulated and thereby facilitate the long-term design of novel therapeutic strategies for treatment of inflammation-linked or neurodegenerative human disorders in which abnormal caspase-1 activation is implicated.
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