Figure 8: Testing predictions of the two-step DISC model.

Molecular models of the inter-molecular DED interactions between FLIP and procaspase 8 (a) and two procaspase 8 molecules (b) that are predicted to occur in the proposed two-step model of DISC assembly. In these models, F122 located on α2 helix of procaspase 8 DED2 is accommodated into a hydrophobic groove formed between α1 and α4 helices of the FLIP or procaspase 8 DED1. (c) Western blot analysis of pull-down experiments using His-tagged FLIP or procaspase 8 DED1/2 as ‘bait’ and Flag-tagged wild-type and mutant full-length and DED-only procaspase 8 as ‘prey’. (d) Assessment of caspase activation induced by rTRAIL in FADD null Jurkat cells transfected with wild-type or mutant Flag-tagged FADD constructs. Twenty-four hours following transfection, cells were treated with 50 ng ml⁻¹ rTRAIL for 6 h prior to assessment of caspase 3/7-like (DEVD-ase) and caspase 8-like (IETD-ase) activity. Mean values and s.d. are shown (n=3); **P<0.01 compared with EV control as determined by t-test. (e) Western blot analysis of DR5 DISC IP carried out in Ren mesothelioma cells overexpressing FLIP(L) or FLIP(S). The DISC was captured 1 h after addition of anti-DR5-beads to cells. (f) Western blot analysis of DR5 DISC IP carried out in HCT116 colon cancer cells transfected with 10 nM control siRNA (SC), FLIP(L)-specific siRNA (FL), FLIP(S)-specific siRNA (FS) or dual FLIP(L)/(S)-targeted siRNA (FT). Cells were transfected for 24 h and co-treated with 10 μM z-VAD-fmk to prevent apoptosis induced by FLIP silencing (z-VAD-fmk does not inhibit processing of p53/55-procaspase 8 to p41/43-caspase 8, but does prevent further processing). The DR5 DISC was captured 1 h after addition of anti-DR5-Beads to cells.