Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells

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.     

Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor

Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one alpha-helix and five beta-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(+/-0.003)x10(-8) M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed.     

Enzymatic active site of caspase-activated DNase (CAD) and its inhibition by inhibitor of CAD

Caspase-activated DNase (CAD) is a deoxyribonuclease that causes DNA fragmentation during apoptosis. In proliferating cells, CAD is complexed with ICAD (inhibitor of CAD) and its DNase activity is suppressed. Here, we established a quantitative assay for CAD DNase that measures the number of 3' hydroxyl groups on the CAD-generated DNA fragments. Chemical modification of histidine residues and substrate protection experiments demonstrated the presence of reactive histidine residues within the active site of the enzyme. Analysis by site-directed mutagenesis suggested that at least four histidine residues in the C-terminal part of the molecule are essential for the catalytic activity of CAD DNase. ICAD did not protect CAD from the chemical modification of the histidine residues, indicating that it does not mask the active site of CAD. In contrast, ICAD blocked the ability of CAD to bind DNA, suggesting that ICAD causes steric or electrostatic hindrance in CAD for substrate DNA. This molecular mechanism for the inhibition of CAD DNase by ICAD is similar to that proposed for colicin endonuclease and its inhibitor, immunity protein.     

Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis

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 suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.     

Multiple feedback loops are key to a robust dynamic performance of tryptophan regulation in Escherichia coli

Internucleosomal DNA fragmentation is an apoptotic event that depends on the activity of different nucleases. Among them, the DNA fragmentation factor B, better known as caspase-activated DNase (CAD), is mainly responsible for this DNA fragmentation in dying cells. CAD is an endonuclease that is chaperoned and inhibited by inhibitor of CAD (ICAD). Activation of CAD needs the cleavage of ICAD by activated caspase-3. During the characterization of the staurosporine-induced apoptotic process in human neuroblastoma cell lines, we have found three novel splice variants of CAD. In all three messengers, the open reading frame is truncated after the second exon of the CAD gene. This truncated open reading frame codifies the CAD protein amino terminal part corresponding to the cell death-inducing DFF45-like effector-N (CIDE-N) domain. We have detected these splicing variants in human tissues and in peripheral white blood cells from 10 unrelated individuals, and their products have been showed to be expressed in certain mouse tissues. We demonstrate that these truncated forms of CAD are soluble proteins that interact with ICAD. We also provided evidences that these CIDE-N forms of CAD promote apoptosis in a caspase-dependent manner.     

Degradation of caspase-activated DNase by the ubiquitin-proteasome system

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin-proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin-proteasome system in the absence of ICAD to maintain protein quality control.     

Multiple domains of DFF45 bind synergistically to DFF40: roles of caspase cleavage and sequestration of activator domain of DFF40.

CAD/DFF40, the nuclease responsible for DNA fragmentation during apoptosis, exists as a heterodimeric complex with DFF45/ICAD. The study presented here augments the accompanying inhibition and chaperone study with an analysis of specific binding strengths and locations of DFF45 binding sites within DFF40. This allows us to show that DFF40/45 interaction is mediated by binding of three functional domains (D1, D2, and D3) of DFF45 to two domains (activator and catalytic) of DFF40. D1 binds exclusively to the activator domain and D2 binds to the catalytic domain of DFF40. Inhibition of DFF40 nuclease activity arises independently from D1 functional sequestration of the activator domain and D2 blockage of the catalytic domain of DFF40. The mechanism of caspase activation of DFF40 is the disruption of the synergistic binding activity of DFF45 domains to DFF40 after caspase recognition and cleavage of DFF45 in the context of a DFF45/40 complex.     

Apoptotic DNA fragmentation

Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die. In this review, the molecular mechanism of and the physiological roles played by apoptotic DNA fragmentation will be discussed.     

Genetic analysis of the short splice variant of the inhibitor of caspase-activated DNase (ICAD-S) in chicken DT40 cells

We have studied the regulation of the caspase-Activated DNase (CAD) by its inhibitor, ICAD. To study the role of ICAD short and long splice forms ICAD-S and ICAD-L, respectively, in vivo, we constructed chicken DT40 cell lines in which the entire coding regions of ICAD alone or ICAD plus CAD were deleted. ICAD and ICAD/CAD double knock-outs lacked both DNA fragmentation and nuclear fragmentation after the induction of apoptosis. We constructed a model humanized system in which human ICAD-L and CAD proteins expressed in DT40 ICAD/CAD double knock-out cells could rescue both DNA fragmentation and stage II chromatin condensation. ICAD-S could not replace ICAD-L as a chaperone for folding active CAD in these cells. However, a modified version of ICAD-S, in which the two caspase-3 cleavage sites were replaced with two tobacco etch virus (TEV) protease cleavage sites (ICAD-S(2TEV)) and which was therefore resistant to caspase cleavage, did inhibit CAD activation upon induction of apoptosis in vivo. Moreover, ICAD-L(2TEV) was functional as a chaperone for the production of active CAD in DT40 cells. In extracts prepared from these cells, we were able to activate CAD by cleavage of ICAD-L(2TEV) with TEV protease under non-apoptotic conditions. Thus, ICAD appears to be the only functional inhibitor of CAD activation in these cell-free extracts. Taken together, these observations indicate that ICAD-S may function together with ICAD-L as a buffer to prevent inappropriate CAD activation, particularly in cells where ICAD-S is the dominant form of ICAD protein.     

Degradation of caspase-activated DNase by the ubiquitin–proteasome system

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin–proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin–proteasome system in the absence of ICAD to maintain protein quality control.     

Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.Structured summary of protein interactionsDrep2 CIDE and Drep-3 CIDE bind by blue native page (View interaction)Drep2 CIDE and Drep-3 CIDE bind by molecular sieving (View interaction)     

Structure of the heterodimeric complex between CAD domains of CAD and ICAD

We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an alpha-helix and a beta-sheet, and are aligned side by side in the complex. Notably, the positive charges on the strand beta2 at one end of the beta-sheet of CAD and negative charges around the opposite end of the beta-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD-ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.     

Erythropoietin activates caspase-3 and downregulates CAD during erythroid differentiation in TF-1 cells - a protection mechanism against DNA fragmentation

The involvement of caspase-3 and its failure in the induction of DNA fragmentation during erythropoiesis were investigated with TF-1 cells. During erythroid differentiation, caspase-3 activation and cleavage of caspase-3 substrates such as ICAD (inhibitor of caspase-activated DNase) were detected without concomitant phosphatidyl-serine (PS) externalization and DNA fragmentation. These observations are in contrast to our understanding that DNA is degraded by CAD (caspase-activated DNase) when ICAD is cleaved by caspase-3. Our study demonstrates that CAD is downregulated at the mRNA and protein level during the erythroid differentiation in TF-1 cells. This provides a mechanism for the first time how cells avoid DNA fragmentation with activated caspase-3.     

Co-translational folding of caspase-activated DNase with Hsp70, Hsp40, and inhibitor of caspase-activated DNase.

CAD (caspase-activated DNase) that causes chromosomal DNA fragmentation during apoptosis exists as a complex with ICAD (inhibitor of CAD) in proliferating cells. Here, we report that denatured CAD is functionally refolded with Hsc70-Hsp40 and ICAD. Hsc70-Hsp40 suppresses the aggregation of the denatured CAD, but cannot restore its enzymatic activity. In contrast, ICAD could not suppress the aggregation of CAD, but supported the CAD's renaturation with Hsc70-Hsp40, indicating that ICAD recognizes the quasi-native folding state of CAD that is conferred by Hsc70-Hsp40. Using an in vitro translation system, we then showed that during CAD translation, Hsc70-Hsp40 as well as ICAD bind to the nascent CAD polypeptide, while on ribosomes. These results indicate that ICAD together with Hsc70-Hsp40 assists the folding of CAD during its synthesis, and that the CAD*ICAD heterodimer is formed co-translationally.     

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.     

Apoptotic nuclear morphological change without DNA fragmentation.

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.     

Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster

DNA fragmentation, a hallmark of apoptosis, is regulated by a specific nuclease called caspase-activated DNase (CAD) and its inhibitor (ICAD). When cell lysates from Drosophila S2 cells were chemically denatured and the denatured proteins were removed after dialysis, the supernatant inhibited Drosophila CAD (dCAD). To identify the inhibitor, we tested recombinant DREP-1, which was previously identified using the Drosophila EST data base and found it also inhibited dCAD DNase. An antibody against DREP-1 inhibited the ICAD activity in the S2 cell extracts, confirming the identification of DREP-1 as a Drosophila homolog of ICAD (dICAD). The recombinant DREP-1/dICAD was cleaved at a specific site by human caspase 3 as well as by extracts prepared from S2 cells undergoing apoptosis. Biochemical fractionation and immunoprecipitation of dICAD from S2 cell extracts indicated that dICAD is complexed with dCAD in proliferating cells. The expression of the caspase-resistant form of dICAD/DREP-1 in a Drosophila neuronal cell line prevented the apoptotic DNA fragmentation. Northern hybridization and the immunohistochemical analyses revealed that the expression of the dICAD gene is developmentally regulated.     

Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis

Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase-activated DFF40/ CAD nuclease, which is chaperoned and inhibited by DFF45/ICAD. CIDE proteins share a homologous regulatory CIDE-N domain with DFF40/CAD and DFF45/ ICAD. Here we report the solution structure of CIDE-N of human CIDE-B. We show that the CIDE-N of CIDE-B interacts with CIDE-N domains of both DFF40 and DFF45. The binding epitopes are similar and map to a highly charged bipolar surface region of CIDE-B. Furthermore, we demonstrate that the CIDE-N of CIDE-B regulates enzymatic activity of the DFF40/ DFF45 complex in vitro. Based on these results and mutagenesis data, we propose a model for the CIDE-N/ CIDE-N complex and discuss the role of this novel bipolar interaction in mediating downstream events of apoptosis.