| Proteolysis is a fundamental process in biology; it plays a crucial role across development of multicellular organisms, aids in maintaining tissue homeostasis, and is integral in cell signaling. Intracellular proteolysis frequently focuses on proteasome mediated protein degradation, however the tightly regulated and selective proteolysis mediated by the cysteine-aspartyl specific proteases, caspases, leave their substrates intact. The growing list of caspase substrates now tops 1500 proteins; a key unmet question is to differentiate how individual substrate cleavages directly lead to the profound morphological transformations that are the hallmark of apoptotic cells. Within we review current approaches for selective activation of apoptosis via activation of caspases by small molecules and genetic approaches. In addition we detail our novel approach that includes an optimized site-specific and inducible split-protein protease to interrogate the role of a classic apoptotic node, the Caspase Activated DNase (CAD). We describe our engineering platform of post-transcriptional gene replacement (PTGR), where-by endogenous bi-allelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to cleavage by our engineered TEV protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines.;Additionally, we show the utility of our technology in deciphering synthetic lethality resulting from coordinated proteolysis of caspase substrates that control the apoptotic hallmark of chromatin fragmentation. The development of a caspase-3 death switch for preclinical apoptotic biomarker discovery is discussed and future preclinical experiments are outlined. The final chapter shifts focus and discusses a protein engineering strategy for conditional control of kinases via split-protein engineering, splitting of a protein kinase (sKin) and chemical induced dimerization. We described a novel approach to selectively activate individual kinase signaling pathways, independent of their upstream signaling nodes. In addition it outlines preliminary phosphoproteomic data that identifies downstream phospho-tyrosine substrates of c-Abl kinase. |
