Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the non-redundant roles of the executioner caspases-3, -6 and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-Tobacco Etch Virus (TEV) protease under small-molecule control, that we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.
However, to study loss of function proteolysis, we have developed a novel bicistronic viral vector system to knock-in engineered substrates, thus enabling loss of function analysis in the cellular context. The targeting vector comprises a microRNA (miRNA) expression cassette directed against the endogenous substrate locus. In this case, we express an engineered caspase substrate with a miRNA directed against the endogenous copy of the substrate of interest. Therefore, co-expression of both cassettes on the same transcript results in (A) the miRNA-mediated knockdown of the endogenous caspase substrate and (B) knock-in of an engineered variant that is susceptible to cleavage by split-TEV, which enables functional analysis of site-specific caspase proteolysis one substrate at a time. This rapid, cell-based engineering of protein alleles we call "Post-Transcriptional Gene Replacement" or PTGR.
To faithfully recapitulate ICAD cleavage and CAD activation, PTGR vector technology is applied to replace endogenous ICAD with a TEV-cleavable allele, ICAD-TevS. In principle, this strategy preserves the chaperone function of ICAD, and CAD activation in cells can then be controlled by the SNIPer in a temporal and dose-dependent fashion. Importantly, we induce CAD cleavage in the absence of caspase activation using conventional apoptotic inducers.