The Role of Pidd Protein in Adenoviral Induction of Apoptosis

The Adenovirus E1A gene sensitizes cells to genetic insults and apoptosis, most notably in response to cytotoxic factors from innate immune cells. The mechanisms by which E1A sensitizes cells to apoptotic stressors have not been fully elucidated, however E1A actively represses NF-κB anti-apoptotic defenses, thereby sensitizing cells to alternative apoptotic cascades. Recent studies alternatively indicate that E1A also actively induces Caspase-2 activation and mitochondrial injury in the presence of certain cytotoxic injuries. Caspase-2 is cleaved and activated in a large complex consisting of p53-inducible protein with a death domain (PIDD), an adaptor protein RAIDD, and pro-caspase 2. PIDD auto-proteolyzes into several fragments and the C-terminal PIDD-CC fragment is required for activation of the PIDDosome in response to cytotoxic stress. We hypothesized that E1A-induced sensitivity and induction of Caspase-2 activity is dependent on PIDD, and in particular PIDD autoproteolysis to the PIDD-CC fragment. A PIDD knockdown was generated in mouse fibroblast cells that also expressed E1A to determine the contribution of PIDD protein to apoptotic sensitivity. A PIDD processing-mutant, incapable of terminal processing to the PIDD-CC fragment, was also generated to establish whether PIDD auto-cleavage is required for E1A-induced sensitivity. We observed that knockdown of PIDD and inhibition of PIDD processing significantly decreased sensitivity of E1A-expressing cells, indicating that PIDD indeed plays a role in the effects of E1A on caspase-2 activation and sensitization to cytotoxic stressors. These results may ultimately contribute to improvements on the application of Adenovirus E1A in anti-cancer therapeutics