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.

CD95-mediated apoptotic and NF-κB signaling were described by a simple kinetic model. We used a model reduction technique to reduce the number of reactions from 92 to 23 while maintaining a good model fit.p43-FLIP, which is generated at the CD95 DISC by procaspase-8 cleavage, was found to be the link between the CD95 DISC and the NF-κB pathway. P43-FLIP interacts with the IKK complex and leads to its activation.The CD95 DISC complex acts as a signal processor that diverges signals into the apoptotic and NF-κB pathways depending on the amounts of specific DISC proteins.Life/death decisions in CD95 signaling are determined by c-FLIPL and procaspase-8 in a non-linear way.

A systems biology–based approach shows that life and death decisions for cells depend on the stoichiometry of c-FLIP isoforms.

Apoptosis is a process common to all multicellular organisms. Apoptosis leads to the elimination of cells via a complex but highly defined cellular programme. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, AIDS and neurodegeneration. Recently, a substantial step forward in understanding the complex apoptotic pathways has been made by utilising systems biology approaches. Systems biology combines rigorous mathematical modelling with experimental approaches in a closed loop cycle for advancing our knowledge about complex biological processes. In this review we describe the contemporary systems biology studies devoted to apoptotic signalling and focus on the question of how systems biology helps to understand life/death decisions made in the cell and to develop new approaches to rational treatment strategies.

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIPL, and the short c-FLIP isoform, c-FLIPR, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.

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