Caspase 8 and MALT1 Exosite Pinpointing Assay

Interest in the allosteric inhibition of proteases has been stimulated by recurring difficulties in the development of highly selective active-site inhibitors. These difficulties are due to the overlap of active-site functionalities, as well as to low inhibitor specificities. This project aims to develop an assay that pinpoints allosteric sites on proteases. I will specifically design an assay for the proteases, caspase-8 and MALT1, both of which have been associated to oncogenesis through recurrent mutations. Caspase 8 is an initiator caspase in the apoptotic pathway, while MALT1 is a caspase-like protease linked to MALT lymphoma. The assay will be based on CYLD, which is a natural protein-substrate for both proteases. Generation of this substrate will be achieved by coupling a D-AFC flourophore to the C-terminus of the protein domain of interest. The Salvesen lab hypothesizes that there is a difference in rates of catalysis between optimal tetrapeptide substrates and natural protein substrates. I will determine and compare the catalytic parameters k_cat and K_m for both substrates via spectrometry; and thereby, verify assay functionality. So far, I have managed to express CYLD in soluble conditions for the flourophore coupling reaction, but the yields of the reaction must be optimized; once this is done, I will proceed with the spectrometry experiments. This new technology will improve our mechanistic understanding of protease-to-substrate interactions, and this in turn will enhance the process of drug discovery.