title: In vitro reconstitution of the interactions in the PIDDosome.
abstract: Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.
authors.affiliation: Department of Biochemistry, School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan, South Korea.
authors.forename: Tae-ho
authors.initials: TH
authors.lastname: Jang
authors.forename: Chao
authors.initials: C
authors.lastname: Zheng
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.forename: Ju-Hong
authors.initials: JH
authors.lastname: Jeon
authors.forename: Hyun Ho
authors.initials: HH
authors.lastname: Park
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053420:CRADD Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D053143:Caspase 2 / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004249:DNA Damage; D053418:Death Domain Receptor Signaling Adaptor Proteins; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D053420:CRADD Signaling Adaptor Protein; C104252:CRADD protein, human; D002352:Carrier Proteins; D053418:Death Domain Receptor Signaling Adaptor Proteins; D046912:Multiprotein Complexes; C485896:PIDD1 protein, human; D053143:Caspase 2