Exploring the Evolution of a Unique Allosteric Regulatory Mechanism in Caspase‐6

Abstract

Caspase‐6 selectively degenerates excess axons during brain development, and then activity is diminished in healthy adult brains. However, it is found expressed and active in all stages of Alzheimer's Disease (AD), so it has been hypothesized to drive AD pathology. However, highly specific caspase inhibitors are necessary for meaningful experimentation, but remain elusive. Caspase active site inhibitors exhibit cross‐reactivity due to structural conservation of the caspase family's active site, which has lead to inconclusive experimental results. We are exploring allosteric options by studying helix‐3 of caspase‐6, in which twelve N‐terminal residues undergo a strand‐to‐helix conformational change that renders the enzyme inactive when zinc binds to a unique exosite. We used a helical propensity prediction algorithm (AGADIR) to design two mutants that prevent the transition; (1) locked in the active canonical conformation, and (2) locked in the inhibited extended helix conformation (EHC). For the first mutant, we redesigned helix‐3 of caspase‐6 to mimic the propensity seen in caspase‐3, because other capases cannot undergo the transition. We found that only two out of the eighteen residues in the extended helix region are conserved, and that only two mutations were necessary to restore the helical propensity found caspase‐3. We will also explore the access to the EHC in other caspases in order to glean information about how the new function of helix‐3 evolved. Our lab's goal is to learn about the intricacies of caspase allosteric regulation in order to bypass the active‐site conservation dilemma. We will then exploit these naturally existing regulatory mechanisms for the advancement of human disease research.