title: Isolation, characterisation and reconstitution of cell death signalling complexes.
abstract: Apoptosis and necroptosis are dependent on the formation/activation of distinct multi-protein complexes; these include the Death-Inducing Signalling Complex (DISC), apoptosome, piddosome, necrosome and ripoptosome. Despite intense research, the mechanisms that regulate assembly/function of several of these cell death signalling platforms remain to be elucidated. It is now increasingly evident that the composition and stoichiometry of components within these key signalling platforms not only determines the final signalling outcome but also the mode of cell death. Characterising these complexes can therefore provide new insights into how cell death is regulated and also how these cell death signalling platforms could potentially be targeted in the context of disease. Large multi-protein complexes can initially be separated according to their size by gel filtration or sucrose density gradient centrifugation followed by subsequent affinity-purification or immunoprecipitation. The advantage of combining these techniques is that you can assess the assembly of individual components into a complex and then assess the size and stoichiometric composition of the native functional signalling complex within a particular cell type. This, alongside reconstitution of a complex from its individual core components can therefore provide new insight into the mechanisms that regulate assembly/function of key multi-protein signalling complexes. Here, we describe the successful application of a range of methodologies that can be used to characterise the assembly of large multi-protein complexes such as the apoptosome, DISC and ripoptosome. Together with their subsequent purification and/or reconstitution, these approaches can provide novel insights into how cell death signalling platforms are regulated in both normal cell physiology and disease.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, PO Box 138, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hughes
authors.forename: Claudia
authors.initials: C
authors.lastname: Langlais
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cain
authors.forename: Marion
authors.initials: M
authors.lastname: MacFarlane
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D053478:Apoptosomes / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D055503:Protein Multimerization; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D053478:Apoptosomes; D053418:Death Domain Receptor Signaling Adaptor Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505653:TNFSF10 protein, human