Abstract
An engineered Fas-associated death domain protein (FADD), 2DEDplusE-made previously by fusing the tandem DEDs of FADD to the E protein of lambda phage-greatly enhances apoptosis-inducing activity in adherent cells in vitro. To investigate the mechanism of apoptosis-inducing activity of this engineered FADD, we compared the apoptosis-inducing activity of various other engineered FADDs. The tandem DED of 2DEDplusE contributed most to the enhancement of apoptosis, and the E protein contributed moderately. The engineered factor produced artificial death-inducing signaling complex (DISC)-like signals in the cytoplasm that appear as grains under fluorescence microscopy. Membrane blebbing associated with apoptosis was observed just after formation of grain-like signals. Immunoprecipitation analysis demonstrated that 2DEDplusE-FLAG can bind p43/p41 forms of caspase 8 but E protein-FLAG cannot. Gel filtration analysis demonstrated that 2DEDplusE forms a large complex containing partially cleaved procaspase 8 (p43/p41) in the cytoplasm; the size of this complex varies greatly. In the absence of an extrinsic signal, the engineered FADD formed artificial DISC in the cytoplasm, and then its tandem DED activated procaspase 8, which in turn executed apoptosis. The engineered FADD complex closely mimicked intrinsic DISC and increased apoptosis-inducing activity.