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.

Figure 1

Kinetics of procaspase-8/-10 cleavage products at the DISC. (a) Schematic representation of procaspase-8 and -10. (b) SKW6.4 cells were stimulated with 1000 ng/ml CD95L for indicated periods of time. CD95 DISC IPs were analyzed using western blot and probed for the indicated proteins. Band intensities of the cleavage products of procaspase-8 and -10 at the DISC were quantified using ImageJ. The amount of each cleavage product for procaspase-8 or -10 was measured in relation to the total amounts of procaspase-8 or -10, respectively. (c) Distribution of procaspase-8 cleavage products measured by quantitative western blot. To determine the distribution of the cleavage products for procaspase-8, the band intensities for the procaspase-8 and its cleavage products at the DISC-IP, i.e., p55/53, p43/p41 and p26/p24, were quantified and divided by the sum of the intensities of all procaspase-8 proteins. The mean±S.D. is shown (n=4). (d) Distribution of procaspase-10 cleavage products measured by quantitative western blot. The mean±S.D. is shown (n=5). (e) Distribution of procaspase-8 cleavage products measured by quantitative mass spectrometry. To determine the distribution of the cleavage products for procaspase-8, the peak areas at the mass spectrometry spectrum for the procaspase-8 and its different cleavage products at the DISC-IP were quantified and divided by the sum of the amounts of all procaspase-8 proteins as in the quantifications using western blot. The mean±S.D. is shown (n=3). Statistical significance: *P<0.05, ***P<0.001

Figure 2

Kinetics of procaspase-8 and -10 in whole cellular lysates. SKW6.4 cells were stimulated with 1000 ng/ml CD95L for different time points and analyzed by western blot using antibodies against procaspase-10 (a), procaspase-8 (b), and actin. 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. Quantification of procaspase-10 (a) and -8 (b) cleavage products (mean±S.D., n≥3, right panels). The samples in (b) were loaded on the same gel, but the blot was cut (marked by black line) for presentation purposes

Figure 3

c-FLIP DED-DED and procaspase-8 prodomain do not block procaspase-8 chain formation. Immunofluorescence microscopy was used to analyze procaspase-8 chain formation by (a–c) overexpression of procaspase-8-mCherry together with c-FLIP DED-DED-GFP (a), caspase-8 prodomain-GFP (b), or MC159-GFP (c) in HeLa-CD95 cells. (d–g) Single overexpression of c-FLIP prodomain-GFP (d), MC159-GFP (e), caspase-8 prodomain-GFP (f), or procaspase-8-mCherry (g) in HeLa-CD95 cells. In (a–c) and (g), 50 μM zVAD-fmk was added at the time of transfection

Figure 4

c-FLIP DED-DED and procaspase-8 prodomain inhibit CD95-induced cell death. (a) CellTiter-Glo assay was performed on HeLa-CD95 cells, which were transfected with procaspase-8 prodomain-GFP, c-FLIP DED-DED-GFP, GFP, or left untransfected (−). The experiments were carried out after 16h expression and always performed with different cell numbers (1250, 2500, 5000, and 10 000) in duplicates. The mean±S.D. is shown (n=3). RLU, relative luminescence units. (b) HeLa-CD95 cells overexpressing the indicated constructs were stimulated with different concentrations of CD95L for 4 h and cell death measured by PI staining and flow cytometry. Cell viability was calculated as described in Materials and methods. The results for GFP-positive cells are shown. (c) Same as in (b), but for GFP-negative cells or untransfected cells (−). The data in (b and c) show mean±S.D. (n=3). Representative FACS stainings can be found in Supplementary Figure S3. (d) Upon overexpression of the procaspase-8 prodomain or short c-FLIP protein (depicted in brown), they could be recruited into the DISC alongside full-length procaspase-8 leading possibly to heterodimer formation. Consequently, procaspase-8 cannot efficiently form homodimers and its activation is inhibited

Figure 5

c-FLIP overexpression does not block procaspase-8 recruitment into the DISC. (a) HeLa-CD95 (12) or HeLa-CD95 c-FLIP_R/L overexpressing cells (FRL) were stimulated with 1 μg/ml anti-APO-1 and CD95 DISC IPs were prepared. Lysates and DISC IPs were separated by SDS-PAGE and analyzed by western blot. The bands marked by an asterisk denote unspecific bands in the anti-caspase-8 (C15) western blot and IgG light chain in the anti-c-FLIP western blot. (b) HeLa-CD95 cells stably overexpressing c-FLIP_R and c-FLIP_L (HeLa-CD95-FRL) or HeLa-CD95 cells were stimulated with different concentrations of Super Fas Ligand (SF-L) for 4h and cell viability was measured by ATP assay (n=3). RLU, relative luminescence units. Statistical significance: **P<0.01, *P<0.05. (c and d) c-FLIP_R does not interfere with FADD self-association. GFP-FADD, Xpress-FADD, or c-FLIP_R was overexpressed in 293T cells either alone or in combination. Cell lysates show equal expression of the two FADD plasmids. (c) Co-expression of both FADD constructs together with c-FLIP_R did not block or reduce co-IP of FADD (d)

Figure 6

Mathematical modeling suggests that DED chains can be regulated by ‘increasing instability'. (a) Model Topology. CD95 is stimulated by CD95L followed by recruitment of FADD. Subsequently, DED proteins, i.e., procaspase-8 (C8), c-FLIP_L (FL), and c-FLIP_R (FR) are recruited resulting in chain formation with different length and composition. Procaspase-8 homodimers are fully processed via p43/p41 (p43) to prodomain (pC8) and p18 (C8*), while procaspase-8/c-FLIP_L heterodimers are partially processed to p43/p41 (p43) and procaspase-8/c-FLIP_R heterodimers are not processed. Fully processed caspase-8 (C8*) is released into the cytosol, but the prodomain (pC8) remains at the DISC. In addition, complete DED chains can be disassembled at any time point. Full-length procaspase-8 (p55/p53) can be degraded following chain disassembly or recruited to other receptors; p43/p41 can be further processed to p18. Free p18 (C8*) or prodomain (pC8) is degraded. Red arrows indicate procaspase-8 processing steps and black arrows other reactions. (b) Simulations of procaspase-8 processing (solid lines) compared with experimental data (points) (mean±S.D.; n⩾3) for p55/p53 (blue), p43/p41 (green), p18 (red), and p26/p24 (purple). Experimental data are taken from Supplementary Figure S4A and our previous work. A full list of parameter values is shown in Supplementary Table S1. (c) Sensitivity analysis performed on the parameters of the model. Each parameter was varied individually from x/5 to x*5, where x was the final value in the simulation (Supplementary Table S1) and the sensitivity to model output was computed. Sensitivities above the average on each state variable upon changing different parameters are indicated using the same color code as in (b). The parameters regulating procaspase-8 association (n_DED) and dissociation (p_Proc8_release) and DISC stability (p_dissociate_DISC) had an above-average effect on multiple state variables at different CD95L levels

Figure 7

Mathematical modeling unravels the inhibitory mechanism of the procaspase-8 prodomain. (a) Distribution of procaspase-8 cleavage products for the indicated CD95L concentrations over time in simulations. (b) Distribution of procaspase-8 cleavage products at the CD95 DISC IP for 20 ng/ml CD95L after 15 min and 50 ng/ml CD95L after 60 min stimulation measured by quantitative mass spectrometry. The mean±S.D. is shown (n=3). Statistical significance: n.s., not significant (P>0.05). (c) Scheme representing negative feedback in procaspase-8 activation. Negative feedback of the procaspase-8 prodomain was introduced into the model by allowing insertion of procaspase-8 into DED chains thus forming inactive heterodimers (right) compared with allowing only recruitment to the end of the DED chain (left). (d) Simulations of procaspase-8 processing (solid lines) with addition of the negative feedback to the model compared with experimental data (mean±S.D.; n⩾3) for p55/p53 (blue), p43/p41 (green), p18 (red), and p26/p42 (purple). Experimental data were taken from Supplementary Figure S4A and our previous work. (e) Distribution of procaspase-8 cleavage products for the indicated CD95L concentrations over time in simulations with negative feedback loop

Figure 8

Model of CD95 DISC dynamics. (a) The suggested role of procaspase-10 in the DED chain. Procaspase-10 is only marginally present compared with procaspase-8 resulting in procaspase-8/-10 heterodimers in the chain, which are most likely not catalytically active. Therefore, procaspase-10 is mainly processed by procaspase-8 activity in the chain leading to the generation of p47/p43 cleavage product. (b) The suggested role of c-FLIP in the DED chain. c-FLIP_L/S/R is recruited to the chain and form heterodimers with procaspase-8. Upon high levels c-FLIP_S/R inhibits procaspase-8 activation in the DISC