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Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD
L M Shearwin-Whyatt1, N L Harvey, S Kumar
1Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, Australia.
Insights
RAIDD protein interacts with caspase-2, localizing to cellular structures that resemble death effector filaments. This CARD-dependent complex formation is crucial for apoptosis execution and may be regulated by RAIDD
Area of Science:
- Cell Biology
- Molecular Biology
- Apoptosis Signaling
Background:
- RAIDD (RIP-associated ICH-1/CLARP-containing protein) is a CARD-domain containing molecule.
- RAIDD interacts with procaspase-2 in a CARD-dependent manner, potentially recruiting it to TNFR1.
- The subcellular localization and complex formation of RAIDD and caspase-2 are not fully understood.
Purpose of the Study:
- To investigate the subcellular localization of RAIDD and its interaction with caspase-2.
- To explore the role of RAIDD's CARD domain in protein complex formation and apoptosis.
- To understand the regulation of RAIDD-CARD oligomerization.
Main Methods:
- Immunofluorescence microscopy to study subcellular localization.
- Co-expression and co-localization studies of RAIDD and caspase-2.
- Analysis of RAIDD-CARD overexpression and filament formation.
Main Results:
- Endogenous RAIDD is primarily cytoplasmic with some nuclear localization.
- Ectopic expression of caspase-2 recruits RAIDD to the nucleus.
- Co-expression of RAIDD and caspase-2 leads to CARD-dependent colocalization into discrete subcellular structures.
- Overexpression of RAIDD-CARD induces filamentous structures via CARD-mediated oligomerization, similar to DED-containing filaments.
- RAIDD-CARD filaments partially co-localize with death effector filaments (DEFs).
Conclusions:
- RAIDD-CARD can form higher-order complexes, analogous to DEDs, which are important for apoptotic execution.
- RAIDD-CARD oligomerization may be regulated by intramolecular folding.
- RAIDD plays a significant role in caspase-2 recruitment and apoptotic signaling pathways.
Abstract:
RAIDD, a caspase recruitment domain (CARD) containing molecule, interacts with procaspase-2 in a CARD-dependent manner. This interaction has been suggested to mediate the recruitment of caspase-2 to the tumour necrosis factor receptor 1 (TNFR1). In this paper we have studied the subcellular localization of RAIDD and its interaction with caspase-2. We demonstrate that endogenous RAIDD is mostly localized in the cytoplasm and to some extent in the nucleus. RAIDD localization is not affected by TNF-treatment of HeLa cells, but in cells ectopically expressing caspase-2, a fraction of RAIDD is recruited to the nucleus. In transfected cells, coexpression of RAIDD and caspase-2 leads to CARD-dependent colocalization of the two proteins to discrete subcellular structures. We further show that overexpression of the RAIDD-CARD results in the formation of filamentous structures due to CARD-mediated oligomerization. These structures were similar to death effector filaments (DEFs) formed by FADD and FLICE death effector domains (DEDs), and partially colocalized with DEFs. Our results suggest that similar to the DED, the RAIDD-CARD has the ability to form higher order complexes, believed to be important in apoptotic execution. We also present evidence that RAIDD-CARD oligomerization may be regulated by intramolecular folding of the RAIDD molecule.