Dimerization mechanisms of two procaspase subfamilies

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DESCRIPTION (provided by applicant): The mechanisms that regulate the activation of procaspases play central roles in the regulation of apoptosis and inflammation. It has been shown that formation of a procaspase dimer is a critical event in maturation. For example, procaspase-1 is thought to be a monomer until interactions in the caspase recruitment domain (CARD) drive dimerization of the protease domains. The scaffold is sufficient to allow autolytic processing. In contrast, we have shown that procaspase-3 is a stable dimer, even though it does not contain a CARD. This suggests different folding and regulatory mechanisms for the activation of procaspases-1 and -3. We hypothesize that differences in the dimer interfaces are the key to whether the protein is a monomer or dimer. In addition, we show that dimerization and enzymatic activity are linked. Based on our protein engineering studies, we hypothesize that the gains in protein stability and enzyme activity are linked via a network of amino acids that extends from the dimer interface to the two active sites. We suggest that procaspases act as molecular machines in which side chain movements in the dimer interface affect the movements in the active site, allowing the substrate-binding pocket to form. We will approach this problem by addressing three key questions in the following specific aims. 1. Do procaspases-1 and -3 fold and assemble via similar mechanisms? Established biophysical methods will be employed to determine the oligomeric properties of procaspase-1 in order to test the current paradigm. 2. How is dimerization linked to active site formation? Using protein engineering techniques, we will examine the apparent linkage of amino acids at four positions near the dimer interface and active sites that affect proper insertion of the active site loops. 3. How does the pro-domain function in folding and assembly? Evidence is presented that the pro-peptide functions as an intramolecular chaperone. Molecular biological and biophysical studies will be employed to determine the precise mechanism of action. This work has the potential to affect therapeutic strategies for a number of autoimmune diseases, leart disease, and cancers because apoptosis is a common factor to these diseases. Learning to electively manipulate the level of apoptosis may well lead to therapeutic strategies for these diseases.