Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1

Abstract

Apoptosis in metazoans is executed by a group of intracellular proteases named caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. We report here the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation.

Fig. 1.

Apaf-1-mediated caspase activation. (A) Coomassie blue staining of purified horse heart cytochrome c (Cyt.c) and recombinant Apaf-1, pro-caspase-9 (Pro-C9), and pro-caspase-3 (Pro-C3) (1 μg of protein each). (B and C) Western blot of caspase-9 and caspase-3, respectively. Apaf-1 at a final concentration of 20 nM was incubated with cytochrome c (Cyt.c, 100 nM final concentration), procaspase-9 (Pro-C9), and procaspase-3 (Pro-C3) at a final concentration of 50 nM in the absence or presence of 10 μM dATP at 30°C for 1.5 h in a final volume of 20 μl (lanes 1 and 2). In lane 3, Apaf-1 and cytochrome c were preincubated at 30°C for 1 h before the addition of procaspase-9, procaspase-3, and dATP and incubated at 30°C for 1.5 h. The samples were subsequently subjected to SDS/PAGE followed by Western blotting analysis using antibodies against caspase-9 and caspase-3. Cleaved products of procaspase-9 and procaspase-3 are labeled as active caspases. (D) The graph shows the caspase-3 activity measured by fluorogenic caspase-3 substrate. Apaf-1 (20 nM) and cytochrome c (100 nM) were preincubated separately at 30°C for 1 h, then incubated with other components (50 nM procaspase-9, 50 nM procaspase-3, and 10 μM dATP) at 30°C for 1 h in a final volume of 20 μl(Left). Apaf-1 and cytochrome c were incubated together with other components at 30°C for 1 h (Center). Apaf-1 and cytochrome c were preincubated together at 30°C for 1 h, then incubated with dATP and other components at 30°C for 1 h (Right). Fluorogenic caspase-3 substrate was added to each sample after the incubation to measure the substrate cleavage as described in Materials and Methods.

Fig. 2.

Apaf-1 is associated with dATP and hydrolyzes it upon cytochrome c binding. (A) A mixture of dATP and dADP analyzed by LC-MS. (B) LC-MS analysis of aqueous phase after 40 ng of recombinant Apaf-1 protein was extracted with phenol as described in Materials and Methods. A mass peak correlated with dATP was indicated. (C) LC-MS analysis was done as in B, but 40 ng of recombinant Apaf-1 was incubated with 20 ng of cytochrome c for 3 h before extraction with phenol. A mass peak correlated with dADP was indicated. (D) LC-MS analysis of aqueous phase after cytochrome c alone was extracted with phenol.

Fig. 3.

Cytochrome c-stimulated dATP hydrolysis by Apaf-1. Apaf-1 (35 pmol) and cytochrome c (175 pmol) were used in a final volume of 40 μl. Aliquots of Apaf-1 alone (filled red circle), Apaf-1 plus cytochrome c (open red circle), and cytochrome c alone (filled pink circle) were incubated at 30°C for 3 h, and dATP hydrolysis was measured at the indicated time points as described in Materials and Methods. For the other three samples, 10 μM of dATP was added to Apaf-1 alone (filled dark blue circle), Apaf-1 plus cytochrome c (open dark blue circle), or cytochrome c alone (filled light blue circle) 2 h after the starting incubation. The dATP hydrolysis was measured by a Malachite Green Phosphate Assay kit as described in Materials and Methods.

Fig. 4.

Apoptosome contains exogenously added dATP. (A) Indicated samples were subjected to glycerol gradient separation after incubation as described in Materials and Methods. Fractions from the glycerol gradient were subjected to SDS/PAGE followed by Western blotting using anti-Apaf-1 antibody. (B) The same glycerol gradient fractions as in A were subjected to caspase-3 activity assay. Aliquots (14 μl) of glycerol gradient fractions were incubated with 50 nM procaspase-9, 50 nM procaspase-3, 10 μM dATP, 100 nM cytochrome c, and 10 μM fluorogenic DEVD caspase substrate in a final volume of 20 μl. Samples were mixed in the test tubes and transferred to 384-well microplates, and caspase-3 activity was measured by using the XFluor4 spectrometry reader (Tecan). (C) dATP incorporation was measured by using [α-³³P]dATP. A total of 10 μCi of [α-³³P]dATP plus 10 μM of dATP were incubated with Apaf-1 and cytochrome c at 30°C for 3 h (blue diamond). For another sample, 10 μCi of [α-³³P] dATP plus 10 μM of dATP was added to Apaf-1 and cytochrome c mixture after they were preincubated for 1.5 h. The sample was incubated for additional 1.5 h before being subjected to glycerol gradient (pink square). The fractions were then collected as in A, and a 4-μl aliquot of each fraction was counted for radioactivity by a liquid scintillation counter. (D) For the active apoptosome, 10 ng of Apaf-1, 5 ng of cytochrome c, and 10 μCi of [α-³³P]dATP plus 10 μM of dATP were incubated at 30°C for 3 h before being subjected to a Superpose 6 chromatography column as described in Materials and Methods. For the inactive apoptosome sample, 10 ng of Apaf-1 and 5 ng of cytochrome c were preincubated at 30°C for 1.5 h. Then, 10 μCi of [α-³³P]dATP plus 10 μM of dATP were added to the sample, and incubation was continued for 1.5 h before the sample was subjected to the Superpose 6 gel-filtration column. An aliquot of 3 μl of peak Apaf-1 fraction was spotted on a TLC plate and analyzed as described in Materials and Methods.

Fig. 5.

Model of apoptosome formation. Apaf-1 is associated with dATP. Upon cytochrome c binding, Apaf-1 hydrolyzes dATP. If there is extra dATP/ATP, dADP is exchanged with dATP and Apaf-1 forms the active apoptosome. When Apaf-1 is incubated with cytochrome c without extra dATP/ATP, dADP-bound Apaf-1 forms the inactive aggregate.