Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation

Abstract

Assembly of a death-inducing signaling complex is a key event in the extrinsic apoptotic pathway, enabling activation of the caspase cascade and subsequent cell death. However, the molecular events governing DISC assembly have remained largely elusive because of the lack of information on mechanism and specificity regulating the death effector domain (DED)-DED interaction network. Using molecular modeling, mutagenesis, and biochemical and ex vivo experiments, we identified the precise binding interface and hot spots crucial for intermolecular DED chain assembly. Mutation of key interface residues (Leu42/Phe45) in procaspase-8 DED-A completely abrogated DED chain formation in HEK293 cells and prevented its association with FADD. A significant 2.6-3.6-fold reduction in procaspase-8 activation was observed in functional cell-death assays after substitution of the interfacial residues. Based on our results we propose a new model for DISC formation that refines the current understanding of the activation mechanism. Upon stimulation, FADD self-associates weakly via reciprocal interaction between helices α1/α4 and α2/α3 of the DED to form an oligomeric signaling platform that provides a stage for the initial recruitment of procaspase-8 through direct interaction with α1/α4 of DED-A, followed by sequential interaction mediated by helices α2/α5 of DED-B, to form the procaspase-8 DED chain that is crucial for its activation and subsequent cell death.

Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death

“…It has been previously demonstrated that the HPV18 E2 TAD binds directly to the tandem DED of procaspase-8 to induce apoptosis 13 . Since isolated procaspase-8 DEDs demonstrate strong tendency to aggregate and exhibit lack of solubility in buffers with different salt concentration and detergents 21 22 , we developed a bacterial co-expression system for interaction analyses. The individual genes, procaspase-8 tandem DEDs, DED-A, DED-B and E2 TAD, were sub-cloned in two compatible plasmid sets with different origin of replication, antibiotic selection, inducer and affinity-tag.…”

“…3A . Recently, we reported that two conserved hydrophobic patches ‘LXXϕ’ and ‘FL’-motifs on the opposite surfaces of DED-A and DED-B, respectively are essential for DED-AB self-association and DEF formation 22 . Importantly, the FL-motif on DED-B corresponds to residues F122/L123, the patch that we identified to be involved in E2-procaspase-8 interaction (see Fig.…”

“…These observations possibly explain as to why we found it difficult to achieve complete abrogation of this interaction by simple deletion or point mutagenesis. Homotypic interactions among the death-fold domain proteins typically involve characteristic helix/helix - helix/helix association, where FL-motif from one DED is accommodated in the hydrophobic groove between α1 and α4 helices of another 22 29 . Our data suggests that interaction of E2 with procaspase-8 mimics homotypic DED-DED complex formation where the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) rests in the groove formed between α2/α3 helices of E2 TAD.…”

“…Thus, these punctate structures may represent oligomerization of caspase-8 that has been induced upon interaction with E2, resulting into caspase activation and hence apoptosis. Recently, we reported the mechanism of classical FADD-procaspase-8 interaction and DISC formation, where FADD interacts with DED-A domain of procaspase-8 to form DED-chain crucial for caspase-8 activation 22 . Since E2 and FADD binding sites on procaspase-8 are distinct, it could be possible that FADD-mediated apoptosis occurs simultaneously or they may be mutually exclusive.…”

Assembly of platforms for signal transduction in the new era: dimerization, helical filament assembly, and beyond

“…3d). Both DED1 and DED2 in the caspase-8 tandem DEDs have an FL motif; it has been established that the FL motif in DED2 is critical for dimerization, oligomerization, and the further activation of caspase-8, whereas the FL motif in DED1 is not associated with either oligomerization or caspase activation [72][73][74] . This result explains the reason that the FL motif in DED2 is critical for caspase-8 activation, showing that it is the primary mediator for caspase-8 domain swapping and dimerization (Fig.…”