Document Detail


Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells.
MedLine Citation:
PMID:  16204257     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Delphine Lechardeur; Sam Dougaparsad; Csilla Nemes; Gergely L Lukacs
Related Documents :
22807997 - H2o2 production downstream of flt3 is mediated by p22phox in the endoplasmic reticulum ...
9664047 - Localization of endogenous arf6 to sites of cortical actin rearrangement and involvemen...
6203027 - Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cel...
21106617 - Alteration in the radiosensitivity of hela cells by dichloromethane extract of guduchi ...
3061597 - Cytodifferentiation of ovarian follicle cells during oocyte growth and maturation.
17721787 - Up-regulation of stress-inducible genes in tobacco and arabidopsis cells in response to...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-10-03
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  280     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-11-28     Completed Date:  2006-02-03     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  40216-25     Citation Subset:  IM    
Affiliation:
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:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
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:
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


Previous Document:  Dissociation of emerin from barrier-to-autointegration factor is regulated through mitotic phosphory...
Next Document:  Does implementation of the European guidelines based on the SCORE model double the number of Norwegi...