Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

Abstract

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

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). …”

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

“…Of note, complex formation 1 hr post-doxorubicin treatment was independent of any apoptotic activity, which was, however, observed between 4 hr (PARP cleavage) and 18–24 hr (caspase-3 cleavage) post-doxorubicin treatment (Figures 6F and 6G, blue boxes). Importantly, absolute quantification of caspase-8, FADD, c-FLIP, and RIPK1 in caspase-8 immunoprecipitates was performed 1 hr post-doxorubicin treatment by a mass spectrometry-based AQUA method (Schleich et al., 2015). This revealed a significant amount of FADD and RIPK1 in complex with caspase-8 in doxorubicin-treated cells (Figure S7J).…”

“…Tryptic digestion was performed by addition of 0.5 μg Trypsin (Trypsin Gold, Promega) and incubated at 37°C for 24 h. AQUA peptides for caspase 8, FADD, c-Flip and RIPK1 were spiked into the digestion solution in an absolute amount of 50 fmol of each peptide as described previously (Schleich et al., 2015). Sequences of AQUA peptides could be received upon request.…”