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Shigekazu Nagata - One of the best experts on this subject based on the ideXlab platform.
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Chapter Fourteen Nucleases in Programmed Cell Death
Methods in Enzymology, 2008Co-Authors: Kohki Kawane, Shigekazu NagataAbstract:Abstract DNA degradation is one of the hallmarks of programmed cell death, or apoptosis. Recent analyses of this process revealed that apoptotic DNA degradation is mediated by two independent mechanisms. First, the caspase‐activated DNase (CAD) cell autonomously cleaves DNA into nucleosomal units in dying cells. Then, after the apoptotic cells are engulfed by macrophages, the fragmented DNA is further degraded by DNase II in the lysosomes of the macrophages. This chapter describes assay procedures for CAD and DNase II. It includes biochemical methods for quantifying DNase activity and cell culture systems to follow cell‐autonomous and noncell‐autonomous DNA degradation. These techniques are useful for studying DNases that are involved in programmed cell death and for following the engulfment of apoptotic cells by phagocytes.
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Degradation of chromosomal DNA during apoptosis
Cell Death & Differentiation, 2003Co-Authors: Shigekazu Nagata, Hiroko Nagase, Kohki Kawane, Naomi Mukae, Hidehiro FukuyamaAbstract: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.
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Mutually regulated expression of Caspase-Activated DNase and its inhibitor for apoptotic DNA fragmentation.
Cell Death & Differentiation, 2003Co-Authors: Hiroko Nagase, Hidehiro Fukuyama, Masato Tanaka, Kohki Kawane, Shigekazu NagataAbstract:Mutually regulated expression of Caspase-Activated DNase and its inhibitor for apoptotic DNA fragmentation
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Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation.
Nature Immunology, 2003Co-Authors: Kohki Kawane, Hiroko Nagase, Hidehiro Fukuyama, Hideyuki Yoshida, Yoshiyuki Ohsawa, Yasuo Uchiyama, Kazuhisa Okada, Tetsuya Iida, Shigekazu NagataAbstract:Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-Activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD−/− thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II−/−CAD−/− embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-β gene was strongly up-regulated in the thymus of DNase II−/−CAD−/− embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.
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Co-translational folding of Caspase-Activated DNase with Hsp70, Hsp40, and inhibitor of Caspase-Activated DNase.
Journal of Biological Chemistry, 2001Co-Authors: Hideki Sakahira, Shigekazu NagataAbstract:Abstract 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 thequasi-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.
Bharathi P Salimath - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of inhibition of ascites tumor growth in mice by curcumin is mediated by NF-kB and caspase activated DNase.
Molecular and cellular biochemistry, 2020Co-Authors: Madesh Belakavadi, Bharathi P SalimathAbstract:One of the most clinically relevant biological activities of curcumin is its anti-cancer property, implicating multiple intracellular pathways in the process. In the present report, we investigated the effect of curcumin on the activation of apoptotic and anti-angiogenic pathways in Ehrlich Ascites Tumor (EAT) cells. Treatment with curcumin in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that the induction of apoptosis in EAT cells showed nuclear condensation, DNA fragmentation and translocation of Caspase-Activated DNase (CAD) to nucleus upon curcumin treatment. Curcumin-induced apoptosis is mediated through activation of caspase-3, which is specifically inhibited by the caspase-3 inhibitor, Ac-DEVD-CHO. On the other hand, the decreased secretion of ascites by EAT cells is corroborated by reduction in VEGF secretion upon curcumin treatment. Further, CD31 immunohistological staining of peritoneum sections in curcumin-treated mice suggests its efficacy in acting as anti-angiogenic compound in EAT cells by inhibiting proliferation of endothelial cells in mouse peritoneum. However, immunoflurescence studies of NF-kB revealed that the inhibition of nuclear translocation of NF-kB p65, a transcription factor required for VEGF gene expression, in curcumin-treated EAT cells. These results suggest a further possible clinical application of this diet-derived compound curcumin, as both proapoptotic and anti-angiogenic compound in association with conventional chemotherapeutic agents.
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Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kB and Caspase activated DNase in ascites tumor model
Biochimie, 2006Co-Authors: A V Deepak, Bharathi P SalimathAbstract:Abstract We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40–50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.
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Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model.
Biochimie, 2005Co-Authors: A V Deepak, Bharathi P SalimathAbstract:We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.
Atsushi Muraguchi - One of the best experts on this subject based on the ideXlab platform.
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Heat shock protein 70 binds Caspase-Activated DNase and enhances its activity in TCR-stimulated T cells.
Blood, 2003Co-Authors: Hiroyuki Kishi, Kenzo Ohtsuka, Atsushi MuraguchiAbstract:DNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the Caspase-Activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)–induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3–activated CAD in a cell-free system. Peptide-binding domain–deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)–binding domain–deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.
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Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes
Journal of Immunology, 2000Co-Authors: Takuya Nagata, Hiroyuki Kishi, Tomoyasu Yoshino, Tadashi Matsuda, Kimie Murayama, Kazuhiro Tsukada, Atsushi MuraguchiAbstract:TCR engagement of immature CD4 + CD8 + thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of Caspase-Activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of Caspase-Activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active Caspase-Activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.
Hideki Sakahira - One of the best experts on this subject based on the ideXlab platform.
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Co-translational folding of Caspase-Activated DNase with Hsp70, Hsp40, and inhibitor of Caspase-Activated DNase.
Journal of Biological Chemistry, 2001Co-Authors: Hideki Sakahira, Shigekazu NagataAbstract:Abstract 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 thequasi-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.
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Enzymatic active site of Caspase-Activated DNase (CAD) and its inhibition by inhibitor of CAD.
Archives of Biochemistry and Biophysics, 2001Co-Authors: Hideki Sakahira, Yasutaka Takemura, Shigekazu NagataAbstract:Abstract 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.
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Identification and developmental expression of inhibitor of Caspase-Activated DNase (ICAD) in Drosophila melanogaster.
Journal of Biological Chemistry, 2000Co-Authors: Naomi Mukae, Hideki Sakahira, Hideki Yokoyama, Takakazu Yokokura, Yasuhiko Sakoyama, Shigekazu NagataAbstract:Abstract 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 thedICAD gene is developmentally regulated.
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A novel activation mechanism of Caspase-Activated DNase from Drosophila melanogaster.
Journal of Biological Chemistry, 2000Co-Authors: Hideki Yokoyama, Hideki Sakahira, Akihiro Iwamatsu, Naomi Mukae, Katsuya Okawa, Shigekazu NagataAbstract:Abstract Caspase-Activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M r of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophilainhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)2(p20)2 complex in gel filtration. When aDrosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD,Drosophila CAD must be cleaved by caspases to be activated.
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Structure of the CAD domain of Caspase-Activated DNase and interaction with the CAD domain of its inhibitor
Journal of Molecular Biology, 2000Co-Authors: Koichi Uegaki, Shigekazu Nagata, Hideki Sakahira, Noboru Yumoto, Takanori Otomo, Masato Shimizu, Yoshimasa Kyogoku, Toshio YamazakiAbstract:Abstract 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 α-helix and five β-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) × 10 −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.
Jun Chen - One of the best experts on this subject based on the ideXlab platform.
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Caspase-Activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures
The Journal of Neuroscience, 2001Co-Authors: R. Anne Stetler, Roger P. Simon, Tetsuya Nagayama, Steven H. Graham, Jun ChenAbstract:Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding Caspase-Activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8–72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.
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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
Journal of Biological Chemistry, 2000Co-Authors: Dexi Chen, R. Anne Stetler, Cristine O'horo, Jun ChenAbstract:Abstract 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/ICADS) 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/ICADS 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/ICADS is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.