ABSTRACT
Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.
Subject(s)
Apoptosis , Caspase 8/metabolism , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Cell Line, Tumor , Enzyme Activation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Jurkat Cells , Mass Spectrometry/methods , Models, Biological , Models, Molecular , Molecular Conformation , Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , fas Receptor/chemistryABSTRACT
Cell death is critical to the normal functioning of multi-cellular organisms, playing a central role in development, immunity, inflammation, and cancer progression. Two cell death mechanisms, apoptosis and necroptosis, are dependent on the formation of distinct multi-protein complexes including the DISC, Apoptosome, Piddosome and Necrosome following the induction of cell death by specific stimuli. The role of several of these key multi-protein signalling platforms, namely the DISC, TNFR1 complex I/II, the Necrosome and Ripoptosome, in mediating these pathways will be discussed, as well as the open questions and potential therapeutic benefits of understanding their underlying mechanisms.
