title: 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.
authors.affiliation: Integrated Biophysics and Structural Biology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Navi Mumbai, India.
authors.forename: Nitu
authors.initials: N
authors.lastname: Singh
authors.affiliation: Integrated Biophysics and Structural Biology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Navi Mumbai, India.
authors.forename: Ali
authors.initials: A
authors.lastname: Hassan
authors.affiliation: Integrated Biophysics and Structural Biology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Navi Mumbai, India kbose@actrec.gov.in.
authors.forename: Kakoli
authors.initials: K
authors.lastname: Bose
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053181:Caspase 8 / Q000378:metabolism*; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D053200:Fas-Associated Death Domain Protein; C505530:CASP8 protein, human; D053181:Caspase 8
keywords: DED chain; FADD; activation; apoptosis; hot spot