Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.

Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.