Determine an Accurate Model for Calcium-Dependent Activation of a Type I Metacaspase from the Fungus Schizophyllum commune

Apoptosis or programmed cell death (PCD) is a cellular process that functions to eliminate damaged cells from an organism. Extensive study in metazoans or vertebrates has indicated that caspase proteins play a prominent role in the initiation of PCD. Unlike metazoans or vertebrates, caspase genes are absent in plants, fungi, and protozoa. Instead, these organisms contain metacaspases which are homologous to caspases in structure, sequence, and function. Initial research has suggested that metacaspases share a similar role to caspases where their proteolytic activity aids in the initiation of PCD. However, unlike caspases, metacaspases are unique in that they can only be activated by the presence of calcium ions. Thus, determining an accurate model for calcium-dependent metacaspase activation could provide insight into the methods behind protease-dependent cell death leading to further strategies to regulate fungal infections. Although it was previously determined that S. commune Type I metacaspases (ScMCA-Ia) are dependent on calcium for activation, the molecular mechanism behind how calcium activates this protease remains unknown. In this work, Differential Scanning Fluorimetry was employed to determine that ScMCA-Ia has two calcium-binding sites with affinities that differ by several orders of magnitude. The high-affinity calcium-binding site is composed of aspartate residues located at positions D279, D295/296, and D326. The low-affinity calcium-binding site is composed of aspartate residues at positions D405 and D411. Interestingly, manganese also binds to ScMCA-Ia and moderately increases activity. Preliminary protein-binding assays indicated that manganese binds to ScMCA-Ia with a binding constant (Kd) similar to that of the low-affinity calcium-binding site. Using this information we propose a model for calcium-dependent metacaspase activation