In vitro reconstitution of the interactions in the PIDDosome.
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.
Jang
Tae-ho
TH
Department of Biochemistry, School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan, South Korea.
Zheng
Chao
C
Wu
Hao
H
Jeon
Ju-Hong
JH
Park
Hyun Ho
HH
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
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