The apoptosome is a large caspase-activating (ϳ700 -1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ϳ135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9 1-130 ) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9 1-130 , demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6 -8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.The morphological and biochemical changes of apoptotic cell death largely result from the activation of a group of cysteine aspartic acid-specific proteases known as caspases (for review see Refs. 1-3). The activation of caspases is a central feature of apoptosis, and key components of this mechanism are highly conserved throughout evolution from Caenorhabditis elegans to Drosophila melanogaster and ultimately to mammals. In C. elegans there are at least four genes, ced-3, ced-4, ced-9, and egl-1, which are critical for the execution of apoptotic cell death. In C. elegans, CED-4 is normally bound to mitochondria and cell membranes by CED-9 (4). Up-regulation of EGL-1 inactivates CED-9, releasing CED-4, which oligomerizes/recruits and induces autocatalytic processing and activation of CED-3 (5). ced-3 encodes for a cysteine protease, which is homologous to interleukin-1␤-converting enzyme or ICE, now known as caspase-1. Thirteen other caspases have now been identified in mammals, some of which are involved in...

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ϳ1.4-MDa and ϳ700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ϳ700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/ HtrA2, active caspase-7 is released from the apoptosome and forms a stable ϳ200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable ϳ200-kDa complex with active caspase-7.The MCF-7 cell line was derived from a patient with metastatic breast cancer and is an often-used model system for studying estrogen receptor-positive breast cancer (for review, see Ref.