Stoichiometry of the CD95 Death-Inducing Signaling Complex: Experimental and Modeling Evidence for a Death Effector Domain Chain Model

Abstract

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.

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

“…The relative abundance of the different cleavage products for procaspase-8 in each slice was quantified by AQUA peptide-based mass spectrometry as described before (Supplementary Figure S2). 29,31 Similar to the western blot quantifications, the relative amounts of the procaspase-8 prodomain p26/p24 at the DISC increased over time (Figure 1e). This quantification further confirmed that the prodomain of procaspase-8a/b p26/p24 is a major part of the DED chains at the CD95 DISC, while the cleavage product p43/p41 is present in lower amounts.…”

“…27,28 Recently, we studied the stoichiometry of the CD95 DISC by AQUA peptide-based mass spectrometry. 29 In combination with western blot analysis supported by mathematical modeling, we revealed an unexpected stoichiometry in which procaspase-8 is more abundant at the DISC compared with FADD. 29 Similar results were obtained for the TRAIL DISC.…”

“…29 In combination with western blot analysis supported by mathematical modeling, we revealed an unexpected stoichiometry in which procaspase-8 is more abundant at the DISC compared with FADD. 29 Similar results were obtained for the TRAIL DISC. 30 Furthermore, the study by Dickens et al 30 has also shown that these chains are formed via DED interactions based on a hydrophobic patch in DED2 of procaspase-8 (F122/L123), referred to as FL-motif, which is highly conserved for procaspase-8, procaspase-10, and c-FLIP (Supplementary Figure S1).…”

“…It was reported that procaspase-8 forms DED chains at the DISC. 29,30 To get more insight into the composition of the DED chains at the DISC, we analyzed CD95 DISCs after immunoprecipitation (IP) by quantitative western blot and mass spectrometry. As in our previous study, we used the B lymphoblastoid cell line SKW6.4.…”

“…Due to the low amounts of procaspase-10 at the DISC, 29 probably only very few procaspase-10 molecules can be found in each chain. Indeed, if we consider the chain length of eight DED proteins, as proposed in our previous study, then this would result in one procaspase-10 per chain at most.…”