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This protocol describes an in vitro model for studying the mechanisms of caspase activation and native apoptosome complex assembly in cell-free extracts. Active caspases in dATP-activated lysates are detected by fluorimetry using a tetrapeptide substrate (DEVD) tagged with a fluorophore (AFC), which, when released, produces a real-time readout for caspase-3 and -7 (DEVDase) activity. Gel filtration is used to isolate the apoptosome complex from the activated lysates, and assembly of Apaf-1 and caspase-9 from their monomeric forms into the multiprotein apoptosome can be confirmed via western blot. Apoptosome complex activity can be shown by incubation with exogenous procaspase-3 and -7 followed by fluorimetric bioassay (to confirm functionality of the processed effector caspases) and/or western blotting (for detection of cleaved caspase-3 and -7). A method for preparation of free procaspases for the bioassay is also described., (© 2015 Cold Spring Harbor Laboratory Press.)

Apoptosis is a highly regulated process that can be initiated by activation of death receptors or perturbation of mitochondria causing the release of apoptogenic proteins. This results in the activation of caspases, which are responsible for many of the biochemical and morphological changes associated with apoptosis. Caspases are normally inactive and require activation in a cascade emanating from an "initiator" or activating caspase, which in turn activates a downstream or "effector" caspase. Activation of initiator caspases is tightly regulated and requires the assembly of caspase-9 (via mitochondrial perturbation) or caspase-8/10 (via death receptor ligation) activating complexes, which are termed the apoptosome and the death-inducing signaling complex (DISC), respectively. These large multiprotein complexes can initially be separated according to size by gel filtration chromatography and subsequently analyzed by affinity purification or immunoprecipitation. The advantage of combining these techniques is one can first assess the assembly of individual components into a multiprotein complex, and then assess the size and composition of the native functional signaling platform within a particular cell type alongside a biochemical analysis of the enriched/purified complex. Here, we describe various methods currently used for characterization of the apoptosome and DISC., (© 2015 Cold Spring Harbor Laboratory Press.)