Full-length Fas-associated Death Domain Protein Interacts with Short Form of Cellular FLICE Inhibitory Protein

摘要

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins.Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8.It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner,through its interaction with FADD and/or caspase-8.However,the interaction of FADD and c-FLIPs (short form) in apoptosis signaling has been controversially discussed.We show the purification and the characterization of human full-length FADD and c-FLIPs expressed in Escherichia coli.The purified FADD and c-FLIPs are shown as homogeneity,respectively,in SDS-PAGE analysis and light-scattering measurements.The folding properties of the alpha-helical structure of FADD and the super-secondary structure of c-FLIPs proteins were characterized by circular dichroism spectroscopy.Furthermore,we report here a series of biochemical and biophysical data for FADD-c-FLIPs binding in vitro.The binding of both FADD and c-FLIPs proteins was detected by BIAcore biosensor,fluorescence measurement,and size-exclusion column (SEC).