| Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells. | |
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MedLine Citation:
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PMID: 16204257 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death. |
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Authors:
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Delphine Lechardeur; Sam Dougaparsad; Csilla Nemes; Gergely L Lukacs |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2005-10-03 |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 280 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 2005 Dec |
Date Detail:
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Created Date: 2005-11-28 Completed Date: 2006-02-03 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: United States |
Other Details:
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Languages: eng Pagination: 40216-25 Citation Subset: IM |
Affiliation:
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Program in Cell and Lung Biology, Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Apoptosis* COS Cells Caspase 3 Caspases / metabolism Cell Line Cercopithecus aethiops Chromatography Chromatography, Gel DNA Fragmentation Deoxyribonucleases / chemistry*, metabolism Dimerization Enzyme Activation Fluorescent Antibody Technique, Indirect Green Fluorescent Proteins / metabolism Hela Cells Humans Immunoblotting Immunoprecipitation Mutagenesis Plasmids / metabolism Protein Binding Protein Biosynthesis Protein Folding Protein Structure, Tertiary Transfection |
| Chemical | |
Reg. No./Substance:
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147336-22-9/Green Fluorescent Proteins; EC 3.1.-/DFFB protein, human; EC 3.1.-/Deoxyribonucleases; EC 3.4.22.-/CASP3 protein, human; EC 3.4.22.-/Caspase 3; EC 3.4.22.-/Caspases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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