Derek MacPherson

Derek MacPherson

Hardy Group

Caspase‑7 with Reprogrammed Specificity Allows Identification of Exosites for Substrate

Recognition

4:00PM

Caspases are a class of cysteine aspartic proteases essential for apoptosis or programmed cell death, a process necessary for maintaining cellular homeostasis and differentiation. There are 12 human caspases, of which 7 are involved in the apoptotic cascade belonging to two distinct subsets, the initiators (casp-2, -8, -9, -10) and executioners (casp-3, -6, -7). Disruption of caspase function in apoptosis has been implicated in cancer, neurodegeneration and inflammation, emphasizing the need to deconvolute individual caspase function, regulation and molecular substrates. Adding to the complexity caspases have similar active-sites composed of four highly flexible loops and recognize similar peptide-length substrates. Despite this similarity, each of the caspases exhibits extraordinary distinguishing power for a particular subset of intracellular targets, the means of which remain unclear. Understanding the molecular details of how each caspase recognize their substrates would provide a new layer of biology to exploit for the development of caspase-specific modulators. Quinary structure is the fifth level of protein architecture, consisting of a series of repulsive and attractive forces that stabilize and influence protein structure and function within the context of natural molecular crowding observed within the cell. One hypothesis is that caspases utilize these forces via unique distal patches, known as exosites, to differentiate and guide their individual substrate interactions. Due to the implications of caspase-6 in neurodegenerative disorders (i.e. Alzheimer’s, Huntington’s) we aimed to generate an evolved specificity caspase (esCasp) that would allow us to distinguish the role of exosites in caspase-6 substrate recognition. esCasp proteins were generated by saturation mutagenesis at critical substrate binding residues in the active site of caspase-7. Utilizing a caged GFP reporter, the library of caspase-7 variants was sorted by their ability to cleave a new recognition sequence (VEID) encoded in the reporter. The end result enabled us to generate esCaspase-7, variants that maintained the body of caspase-7 but had the specificity of the caspase-6. Using N-terminomics a mass spectrometry based proteomics screen, to profile the entire human proteome, we found esCasp-7 displayed the same specificity as the target enzyme, caspase-6, indicating the effectiveness of our evolutionary screen. Because our evolved protease, esCasp-7, has the distal quinary interactions of the parent enzyme caspase-7, and the active-site like caspase-6, it therefore lacks those unique distal features which we hypothesize guide caspase-6 substrate recognition. We predicted that proteins requiring exosites for recognition by caspase-6 would fail to be hydrolyzed by esCasp-7, enabling the first known approach for directly assessing exosite contributions to substrate recognition. We identified lamin A/C as a substrate relying on putative exosite interactions for caspase-6 recognition. Ultimately, the most compelling promise of identifying exosites is their potential for precision medicine, allowing us to uniquely target and exploit caspase-specific exositedirected inhibitors to prevent recognition of one therapeutic substrate, while leaving interactions with all other substrates unchanged. This work provides the basis and the means for the exploration of future substrates related to caspase-6 and neurodegeneration, for which caspase-6-specific exosite inhibitors could be utilized to mitigate the progression of such diseases.