Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate

Abstract

SummaryThe death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex

“…In this recruitment, an interaction surface of FADD that directly contacts Casp-8 is also required for self-association, which may have led to contradictory conclusions (Majkut et al, 2014). Our structural analysis is consistent with the observed hierarchy in DED interactions in which FADD first recruits Casp-8, and Casp-8 further recruits cFLIP (Hughes et al, 2016; Schleich et al, 2016). The predicted comingling of Casp-8 and cFLIP L in the same filament may facilitate the heterodimerization of their caspase domains to promote caspase activation when cFLIP L concentration is relatively low (Figure S7B).…”

“…The assay used recombinant monomeric MBP-cFLIP tDED -Sumo tagged with the TAMRA fluorophore (Figure S6G), which formed filaments upon removal of the N-terminal MBP tag (Figure S6H). In agreement with hierarchical assembly (Hughes et al, 2016; Schleich et al, 2016), the Fas/FADD complex did not significantly enhance cFLIP tDED polymerization, but promoted Casp-8 tDED polymerization under similar conditions (Figure 6F), supporting the weak interaction between FADD and cFLIP.…”

“…Earlier studies have suggested that cFLIP directly interacts with FADD (Majkut et al, 2014) and inhibits Casp-8 activation by competing with Casp-8 for recruitment to FADD (Yang et al, 2005). However, recent studies showed that cFLIP alone only weakly interacted with FADD but assembled effectively into the DISC in a co-operative manner that is dependent on Casp-8, suggesting that DISC assembly is a hierarchical process from FADD to Casp-8, then to cFLIP (Hughes et al, 2016; Schleich et al, 2016). …”

“…The long form of cFLIP, cFLIP L , has an inactive caspase-like domain and hetero-dimerizes with the caspase domain of Casp-8 and cFLIP L has been shown to promote Casp-8 activation (Yu et al, 2009). In contrast, the shorter form of cFLIP, cFLIP S , can inhibit Casp-8 activation (Hughes et al, 2016). Thus cFLIP L and cFLIP S may play differential roles in modulating DR signal transduction.…”

Differential Impacts of Alternative Splicing Networks on Apoptosis

“…Cellular FLICE inhibitory protein (c-FLIP), a caspase-8 homolog, functions as a crucial factor in manipulating apoptotic activity of the Fas/Fas L-mediated pathway [87]. By usage of alternative 5′ splice site within c-FLIP exon 5, the c-FLIP gene generates alternative transcripts which encode three variants, including 55 kDa c-FLIP long (c-FLIP L ), 26 kDa c-FLIP S , and 24 kDa c-FLIP R , in human cells [88].…”