Model for Calcium‐Dependent Activation of a Type I Metacaspase From the Fungus Schizophyllum commune

Abstract

Apoptosis or programmed cell death (PCD) is one of the most important cellular processes which functions to avoid the accumulation of mutations or damage within a cell. PCD has been extensively studied in eukaryotic cells, demonstrating that caspase proteins have a significant role in this process. However, unlike mammalian cells, caspase genes are absent in plants, fungi, and protozoa. Instead, these organisms contain homologous proteins known as metacaspases which have demonstrated similar proteolytic functions in aiding PCD. Determining an accurate model for how these metacaspases are activated could provide insight into the methods behind this protease‐dependent cell death which could lead to further strategies for the regulation of fungal infections. This research focuses on the model of activation for the Type I metacaspases from the fungus Schizophyllum commune. The S. commune Type I metacaspases are dependent on calcium ions for activation; however, the molecular mechanism behind how calcium activates this protease remains unknown.It has been proposed that when calcium binds to the metacapsape, these ions induce a structural rearrangement of the active‐site region leading to activation. To further understand how calcium and other divalent metallic cations influence the activation of this protease, we used kinetic activity assays to quantify metacaspase activity and differential scanning fluorimetry to measure divalent metallic cation‐metacaspase binding.In addition to strong activation by calcium, manganese ions have demonstrated a moderate effect on metaspace activity, while magnesium, copper, samarium, and cobalt don’t affect activity. We will present evidence that calcium binds to two sites on the protein, while manganese binds to only a single site. Furthermore, calcium binding to the Type I metacapses occurs at two sites with affinities that differ by several orders of magnitude. Initial results suggest that it is necessary for calcium to saturate the high affinity site prior to binding to the low affinity site. Thus, it has been predicted that calcium’s initial saturation of the high affinity site induces slight conformation changes to the active‐site; however, once the low affinity site has been saturated, an active‐site loop undergoes further rearrangements resulting in metacaspase activation. Further analysis of binding interactions between the Type I metacapse and calcium (along with other divalent cations) will provide insight into how these structural rearrangements lead to increased metacaspase activity.