Involvement of caspase 3-activated DNase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli (original) (raw)
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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.
Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad)
The Journal of Cell Biology, 2000
Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/ CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporineinduced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.
Journal of Biological Chemistry, 2005
We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIFindependent process.
An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes
Genes & development, 2000
CAD (caspase-activated DNase) can cause DNA fragmentation in apoptotic cells. Transgenic mice that ubiquitously express a caspase-resistant form of the CAD inhibitor (ICAD) were generated. Thymocytes prepared from the mice were resistant to DNA fragmentation induced by a variety of stimuli. However, similar numbers of TUNEL-positive cells were present in adult tissues of transgenic and wild-type mice. Exposure to gamma-irradiation caused a striking increase in the number of TUNEL-positive cells in the thymus of wild-type, but not transgenic, mice. TUNEL-positive nuclei in transgenic mice were confined to thymic macrophages. When apoptotic thymocytes from the transgenic mice were cocultured with macrophages, the thymocytes underwent phagocytosis and their chromosomal DNA underwent fragmentation. This DNA fragmentation was sensitive to inhibitors that block the acidification of lysosomes. Hence, we conclude that the DNA fragmentation that occurs during apoptosis not only can result ce...
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...
Degradation of chromosomal DNA during apoptosis
Cell Death & …, 2003
Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.
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 ...
DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis
Oncogene, 2009
The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.