Dependence of human cell survival and proliferation on the CASP3 prodomain

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled. The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates.