Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems

Abstract

Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.

Keywords: Apaf-1; Apoptosis; Apoptosome; Cell death; Cell-free system; Split-luciferase; Truncated Apaf-1.

Fig. 1

Reconstitution of luciferase activity upon Apaf-1 oligomerization in a cell-free and cell-based systems. (A) The effect of various N/P ratio on transfection efficiency. HEK293 cells were transiently transfected with 2 μg of established plasmids (C-luc Apaf-1 and N-luc Apaf-1) after 24 h with and without 0.5 μM doxorubicin. (B) Time-dependent apoptosis activation in (N/P 12) and 0.5 μM doxorubicin. (C) Apoptosis activation in the absence and presence of different concentrations of doxorubicin. Cells were co-transfected with 2 μg from C-luc and N-luc Apaf-1 (N/P 5) and after 24 h' luciferase activity was assessed. (D) Luciferase activity was evaluated in a mixture of C-luc Apaf-1 and N-luc Apaf-1 extracts with or without dATP and cytochrome c. Activity of C-luc Apaf-1 and N-luc Apaf-1 extracts alone were also tested. Results are the mean ± SD of three separate experiments.

Fig. 2

Gel filtration and immunoblotting analysis of ΔApaf-1. Extracts expressing N-luc ΔApaf-1 and C-luc ΔApaf-1 were mixed 1:1 (0.5 ml from each one) with or without cytochrome c and dATP and then incubated for 15 min at 25 °C and loaded on Sephacryl 300 HR column. The fractions collected, concentrated and detection of N-luc or C-luc ΔApaf-1 constructs was carried out using immunoblot.

Fig. 3

Binding of apoptosome complex to Ni-NTA Agarose Beads. Mixture of N-luc Apaf-1/C-luc Apaf-1; C-luc Apaf-1/N-luc ΔApaf-1 and N-luc Apaf-1/C-luc ΔApaf-1 were prepared, activated with or without cytochrome c and dATP for 15 min at 25 °C and then the mixtures incubated with resin for 1–2 h at 4 °C. Washing was performed in 20 mM imidazole (A) and 165 mM imidazole (B). Fractions were immunoblotted using anti-Apaf-1 monoclonal antibody.

Fig. 4

(A) Different proportions of untransfected and N-luc ΔApaf-1 were prepared, mixed and incubated in the absence and presence of cytochrome c and dATP for 15 min at 25 °C. Then caspase-3 like activity was detected using DEVDA-AMC. (B, C) Procaspase-9 processing without and with dATP/Cc, respectively. (D, E) Procaspase-3 processing in the absence and presence of dATP/Cc, respectively. (F, G) The level of cytochrome c and endogenous Apaf-1 expression in untransfected and N-luc ΔApaf-1 cell extracts, respectively.