A Comprehensive Panel of Turn-On Caspase Biosensors for Investigating Caspase Specificity and Caspase Activation Pathways

Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split‐luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1–10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5. Profiling caspases: A comprehensive panel of genetically encoded turn‐on caspase sensors with a luminescent readout was developed (see figure). The panel of bipartite caspase biosensors was utilized to systematically investigate caspase cleavage specificity profiles. Utilizing this panel of caspase biosensors we identified cross‐talk between inflammatory and executioner caspases.