title: Biochemical Analysis of Initiator Caspase-Activating Complexes: The Apoptosome and the Death-Inducing Signaling Complex.
abstract: 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.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
authors.forename: Claudia
authors.initials: C
authors.lastname: Langlais
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hughes
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cain
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
authors.forename: Marion
authors.initials: M
authors.lastname: MacFarlane
mesh_terms: D017209:Apoptosis; D053478:Apoptosomes / Q000737:chemistry* / Q000378:metabolism*; D053471:Caspases, Initiator / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D053218:Receptors, Death Domain / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D053478:Apoptosomes; D053418:Death Domain Receptor Signaling Adaptor Proteins; D053218:Receptors, Death Domain; D053471:Caspases, Initiator