Uncovering the biological roles of inflammatory caspases through chemical approaches

Project Summary Inflammatory caspases (cysteine-dependent, aspartate specific proteases) are associated with protein complexes termed inflammasomes that play a role in the innate immune response by producing inflammation and cellular death in response to pathogens and danger signals. Dysregulation of the inflammatory response is associated with sepsis and disease states ranging from autoimmune and neurodegenerative disorders to cancer. Due to their role in a wide range of human diseases, inflammatory caspases are a focus for many drug discovery programs. The long term objective for this proposal is the development of tools for assessing inhibition of inflammatory caspases and allowing the role each enzyme in the inflammatory response to be determined. This objective can be best addressed by developing peptide substrates for activity assays that selectively interact with individual inflammatory caspases. To date, the creation of such substrates for inflammatory caspases has been elusive. Prior approaches to generating selectivity have focused on varying the amino acids N-terminal to the aspartate residue. The PIs have shown it is possible to vary the amino acids C-terminal to the aspartate by incorporating the reporting chromophores as side chains of non-natural amino acids and observed markedly different kinetics for an inflammatory caspase reacting with peptides that differed by a single C-terminal amino acid. The mechanism of inflammasome formation is not clear for caspase-4 or -5 and the majority of information available is for caspase-4. In order to resolve the individual biological roles of these enzymes, structural studies of inflammasome formation must be performed on both C-4 and C-5 independently. Previous studies have been performed using inactive mutant enzyme rather than a more biologically relevant active form that is chemically inactivated. The hypothesis of this proposal is that the biological roles and mechanism of activation of each inflammatory caspase can be uncovered by using a chemical approach to studying the activity of each enzyme. The following specific aims will address this hypothesis: 1) Develop and validate peptide substrates with 100- fold selectivity for each inflammatory caspase by varying the amino acids on the C-terminal side of the aspartic acid residue and determine the chromophore pair needed to produce the maximum signal-to-noise ratio. 2) Determine the mechanism of activation for caspase-4 and caspase-5 using mutagenic and chemical methods. The development of selective substrates for inflammatory caspases using multiple, high signal-to-noise dye pairs will allow the activity of each caspase to be assessed via a unique fluorescence reporter. The creation of these substrates will positively impact the field of drug development to combat inflammatory diseases and sepsis. Chemical approaches to studying inflammasome formation will allow the roles of inflammatory caspases in the innate immune response to be de-convolved, contributing to the advancement of our understanding of inflammation. This proposal will provide an interdisciplinary research experience for undergraduate students, exposing them to applications of chemical biology and biophysics in improving human health.