Figure S1. Sequence alignment of procaspase-8, procaspase-10 and c-FLIP prodomains with FADD DED and MC159. The hydrophobic patch (FL-motif) is conserved in all tandem DEDs, which is required for DED interactions. The charge triad motif is highly conserved in c-FLIP. The hydrophobic patch is highlighted by blue boxes, the charge triad motif by yellow boxes. The amino acids, which constitute the charge triad, are marked by arrows.
Figure S2. Experimental workflow for the quantification of the CD95 DISC stoichiometry by AQUA peptide-based mass spectrometry. (A) CD95 DISC IPs from stimulated SKW6.4 cells were separated by 1D SDS PAGE, the gels were sliced into pieces and those slices containing the major DISC proteins were quantified by AQUA peptide-based mass spectrometry (B) Representative quantification of procaspase-8 by mass spectrometry using two different AQUA peptides for p55/p53 (75-50 kDa), p43/p41 (50-37 kDa) and p26/p24 (37-10 kDa).
Figure S3. Procaspase-8 prodomain inhibits CD95-induced cell death. Flow cytometry analysis of GFP (A), procaspase-8 prodomain-GFP (B), c-FLIP DED-DED-GFP (C) transfected or untransfected (D) HeLa-CD95 cells using PI staining. Shown are representative experiments for each condition with and without CD95L stimulation.
Figure S4. Model calibration.(A)Quantification of procaspase-8 prodomain generation kinetics by western blot. SKW6.4 cells were stimulated with 10, 200 and 1,000 ng/ml CD95L, the cells were lysed and total cellular lysates subjected to western blot analysis using an N-terminal procaspase-8 antibody, C-terminal procaspase-8 antibody and actin. Part of thisblot (Stimulation with 1,000 ng/ml) has been also used in Figure 2. These data were combined with our previous quantifications of procaspase-8 processing using western blot for comparison with model simulations in Figure 6B1. One representative experiment of at least three is shown. (B) Quantification of experimental data in Figure 1B obtained by quantitative western blot of procaspase-8 cleavage products distribution at the DISC at 1,000 ng/ml CD95L compared to simulation result (n=4). (C) Western blots of SKW6.4 cells stimulated with 500 ng/ml and 50 ng/ml CD95L. The blots were probed with C-terminal procaspase-8 antibody (C15) and actin. One representative experiment of three is shown.Band intensities were quantified using ImageJ. The intensities of the different cleavage products were normalized to actin and the total amounts of the full-length protein.(D) Simulations of procaspase-8 processing (solid lines) compared to experimental data (mean ± sd; n=3) including p55/p53 (blue), p43/p41 (green) and p18 (red) for 500 ng/ml and 50 ng/ml CD95L. The corresponding representative western blot is shown in panel C(D) Themodelwas evaluated on procaspase-8 processing data obtained in HeLa-CD95 cells upon stimulation with 3µg/ml CD95L, taken from our previous work2. Shown are simulations of procaspase-8 processing (solid lines) compared to experimental data (mean ± sd) for p55/p53 (blue), p43/p41 (green) and p18 (red). Further details can be found in the text and Materials and Methods.
Figure S5. Possible structure of the DED chain. The DED chain was assembled in silicoby ad-hoc structural considerations based on the proposed interaction of DED2 with the FADD DED. Helices α2/α5 (shown in grey) from procaspase-8/10 DED2, comprising the hydrophobic patch, interact with helices α1/α4 (shown in magenta) from FADD DED. The second tandem DED interacts with the first one in the same manner, which results in chain elongation. Due to the structure of the tandem DED and orientation of the interacting helices, the DED might have a helical structure. This might lead to sterical problems with a second DED chain bound to the same FADD molecule via the hydrophobic patch in the FADD DED. The hydrophobic patch in the tandem DEDs is shown in the spheres representation colored by elements showing carbon in gray, oxygen in red and nitrogen in blue. DD = death domain.
Figure S6. Negative feedback of procaspase-8 prodomain. Simulations of procaspase-8 processing with addition of the negative feedback to the model (solid lines) compared to experimental data (mean ± sd; n=3) including p55/p53 (blue), p43/p41 (green) and p18 (red) for 500 ng/ml and 50 ng/ml CD95L in SKW6.4 cells. Experimental data is shown in Figure S4C.
Table S1. Model Parameter values. Parameters were estimatedby parameter estimation using experimental data of procaspase-8 processing in SKW6.4 cells in lysates and at the DISC for 1,000 ng/ml CD95L stimulation. p_DED and p_FADD were calculated based on the distance of a chosen molecule to a specific receptor. This recruitment was repeated n_DED and n_FADD times if recruitment had failed to represent diffusion.*only applied to procaspase-8 molecules released upon disassembly of DED chains. A procaspase-8 dissociation probability (p_Proc8_release) of 0.1 was used without negative feedback, which was slightly reduced upon addition of the negative feedback to 0.07.
Parameter / Value / Parameter / Valuep_C8_deg / 0.02 / processToP18 / 0.05
p_C8_release / 0.7 / processToP43 / 0.16
p_CD95L / 0.2 / p_Proc8_release / 0.1/0.07
n_DED / 43 / p_deg_prodomain / 0.1
n_FADD / 20 / p_dissociate_DISC / 0.02
p_deg_proc8* / 0.5
References
1.Schleich K, Warnken U, Fricker N, Oztürk S, Richter P, Kammerer K, et al.Stoichiometry of the CD95 Death-Inducing Signaling Complex: Experimental and Modeling Evidence for a Death Effector Domain Chain Model. Molecular Cell 2012, 47: 306-319.
2.Fricker N, Beaudouin J, Richter P, Eils R, Krammer PH, Lavrik IN. Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL. The Journal of Cell Biology 2010, 190: 377-389.
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