Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad) (original) (raw)
Related papers
Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells
The Journal of Cell Biology, 2004
Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3–dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the in...
Oncogene, 1999
Degradation of chromosomal DNA into nucleosomesized fragments is one of the characteristics of apoptotic cell death. Here, we examined whether caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs upon exposure to various apoptotic stimuli. When human Jurkat cells were exposed to etoposide, or UV or g radiation, a caspase-3-like protease was activated, and nuclear DNA was fragmented. Human TF-1 cells, which are dependent on granulocyte-macrophage colony-stimulating factor (GM ± CSF), also underwent apoptosis accompanied by the activation of caspase-3-like protease and DNA fragmentation, when cultured without the cytokine. Both Jurkat and TF-1 cells expressed two forms of ICAD, ICAD-L and ICAD-S, which were cleaved upon exposure to these apoptotic stimuli. Among eight dierent caspases examined, recombinant caspases 3 and 7 speci®cally cleaved ICAD synthesized in a cell-free system. An expression plasmid containing mouse ICAD-L mutated at the caspase-3recognition sites was then introduced into Jurkat and TF-1 cells. When the transformants were induced to undergo apoptosis (by treatment with etoposide, UV or g radiation for Jurkat cells, or factor withdrawal for TF-1 cells) they did not show DNA fragmentation, although they still died as a result of these stimuli. These results indicated that CAD, released from ICAD by caspase activation, is involved in the nuclear DNA fragmentation induced by these apoptotic stimuli.
Functional Differences of Two Forms of the Inhibitor of Caspase-activated DNase, ICAD-L, and ICAD-S
Journal of Biological Chemistry, 1999
Caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs during apoptosis. CAD is complexed with an inhibitor of CAD (ICAD) in nonapoptotic, growing cells. Here, we report that mouse WR19L and human Jurkat T lymphoma cells express two alternative forms of ICAD, ICAD-L and ICAD-S, at similar levels. CAD was predominantly associated with ICAD-L in these cell lines. When CAD was expressed alone in Sf9 cells, it was found in insoluble fractions. However, when CAD was co-expressed with ICAD-L and ICAD-S, it was recovered as a soluble protein complexed predominantly with ICAD-L. In vitro transcription and translation of CAD cDNA did not produce a functional protein. Addition of ICAD-L but not ICAD-S to the assay mixture resulted in the synthesis of functional CAD. These results indicated that ICAD-L but not ICAD-S works as a specific chaperone for CAD, facilitating its correct folding during synthesis. Recombinant CAD, as a complex with ICAD-L, was then produced in Sf9 cells. The complex was treated with caspase 3, and CAD was purified to homogeneity. The purified CAD had DNase activity with a high specific activity.
Caspase2 cleaves DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD
Archives of Biochemistry and Biophysics, 2007
To investigate the signal transduction pathway of caspase-2, cell permeable Tat-reverse-caspase-2 was constructed, characterized and utilized for biochemical and cellular studies. It could induce the cell death as early as 2 h, and caspase-2-specific VDVADase activity but not other caspase activities including DEVDase and IETDase. Interestingly, nuclear DNA fragmentation occurred and consistently DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD) was cleaved inside the cell as well as in vitro, suggesting a role of caspase-2 in nuclear DNA fragmentation.
The Journal of biological chemistry, 2000
Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further su...
Oligomerization State of the DNA Fragmentation Factor in Normal and Apoptotic Cells
Journal of Biological Chemistry, 2005
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.
Biochemical and Biophysical Research Communications, 1998
We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant human DNA fragmentation factor 45 kDa subunit (DFF45/ICAD), an inhibitor of the caspase-activated DNase DFF40/CAD, substituted for healthy cytosol in inhibiting DNA fragmentation. An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cytosols but not in the nuclear or membrane fractions of various cultured human cells. Cytosols depleted of DFF45/ICAD by immunoadsorption had little or no inhibitor of nuclear DNA fragmentation activity and no caspase-activated DNA fragmentation activity. We conclude that immunoreactive DFF45/ICAD is the principal inhibitor of apoptotic DNase activity in the cytosol of healthy cells.
Two Distinct Pathways Leading to Nuclear Apoptosis
Journal of Experimental Medicine, 2000
Apaf-1−/− or caspase-3−/− cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1−/− or caspase-3−/− cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in ...