Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Induction of apoptosis by death receptors such as Fas or tumour necrosis factor (TNF) R1 leads to distinct changes in cell morphology, activation of the caspase protease cascade, and the degradation of nuclear chromatin by activated nucleases. Here, we describe the purification and cDNA cloning of a novel 40 kDa endonuclease from Jurkat cells that is activated by caspases. This protein, designated caspase-activated nuclease (CPAN), is sufficient to degrade naked DNA and to induce apoptotic morphology and DNA fragmentation in naive nuclei. CPAN is highly homologous to a recently described mouse nuclease, CAD [1], and may represent the human homologue. Our data on the human cDNA as well as additional data on the mouse homologue suggest that a 30 amino-acid portion of the recently published mouse sequence [1] is incorrect. We show that the activity of human CPAN is regulated by DFF45 [2], an inhibitor necessary for CPAN expression and stabilization in an inactive state in living cells. Proteolytic cleavage of DFF45 by caspases in vitro leads to dissociation of DFF45 fragments from CPAN and activation of CPAN as an endonuclease. CPAN is a tightly regulated endonuclease with unique characteristics that might represent a distinctive family of endonucleases.
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PMID:CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. 956 Mar 46

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.
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PMID:Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis. 1050 74

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.
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PMID:Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. 1052 51

In this paper, we show that there is a two-step process of DNA fragmentation in apoptosis; DNA is first cleaved to large fragments of 50-300 kb that are subsequently cleaved to smaller oligonucleosomes in some, but not all cells. Significantly, only the first stage is considered essential for cell death since some cells, for example human MCF7 breast carcinoma cells and human NT2 neuronal cells, do not show this behavior but still display normal nuclear morphological apoptotic changes. In cells that usually produce small fragments blocking the second (internucleosomal) stage of DNA fragmentation prevents neither nuclear condensation nor apoptosis. We are beginning to understand why the extent of DNA fragmentation during apoptosis varies enormously and why it appears to be a function of the cell type not the inducer. Presumably, this reflects the content of not only endonuclease activit(ies) but also on the ability of the cells to activate caspases, particularly caspase-3, and other proteases that may be involved in endonuclease activation. Since NT2 cells activate caspase-3, but do not correctly process DFF45, other factors must also impinge on the inevitability of that process.
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PMID:Neither caspase-3 nor DNA fragmentation factor is required for high molecular weight DNA degradation in apoptosis. 1066 63

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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PMID:Enzymatic active site of caspase-activated DNase (CAD) and its inhibition by inhibitor of CAD. 1136 Nov 46

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.
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PMID:Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation. 1169 7

A variety of endonucleases has been implicated in apoptotic DNA fragmentation. DNA fragmentation factor (DFF) is one of the endonucleases responsible for DNA fragmentation. Since an oligonucleosomal DNA ladder is not induced in apoptotic Molt-4 cells, we investigated whether or not the absence of ladder formation is related to an inability of DFF endonuclease in the cells. Semiquantitative RT-PCR analysis showed that the mRNA level of DFF-40 and DFF-45 in Molt-4 cells was approximately the same, compared with in other cells, which exhibit different levels of the fragmentation in apoptosis. When Molt-4 cells were induced to undergo apoptosis by neocarzinostatin (NCS) treatment, both caspase-3 activation and DFF-45 cleavage were observed. Furthermore, DFF immunoprecipitated from Molt-4 cells exhibited DNA degradation activity. These results suggest that functional expression of DFF is not sufficient for the induction of DNA fragmentation in Molt-4 cells.
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PMID:Activation of caspase-3, proteolytic cleavage of DFF and no oligonucleosomal DNA fragmentation in apoptotic Molt-4 cells. 1187 77

The sequential generation of large-scale DNA fragments followed by internucleosomal chromatin fragmentation is a biochemical hallmark of apoptosis. One of the nucleases primarily responsible for genomic DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD). DFF40/CAD is a magnesium-dependent endonuclease specific for double stranded DNA that generates double strand breaks with 3'-hydroxyl ends. DFF40/CAD is activated by caspase-3 that cuts the nuclease's inhibitor DFF45/ICAD. The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. However, the nuclease hypersensitive sites can be detected and DFF40/CAD is potentially involved in large-scale DNA fragmentation as well. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis.
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PMID:The DFF40/CAD endonuclease and its role in apoptosis. 1199 94

We investigated the mode of cell death induced by the anthracyclines, aclarubicin, doxorubicin and daunorubicin in the human leukemia cell lines, HL60 and Jurkat. The cells were incubated with drug concentrations up to 500 nM for periods between 3 and 24 hours, followed by morphological and biochemical analyses. All three substances induced DNA fragmentation, evident as DNA laddering and appearance of cells with hypodiploid DNA content, externalization of phosphatidyl serine, activation of caspases and degradation of the apoptosis-specific endonuclease inhibitor DFF45. However, concentrations and times necessary for these effects to occur were different, aclarubicin being the quickest acting drug with a lag phase of 3 h, followed by daunorubicin with 6 h and doxorubicin with 24 h. More importantly, aclarubicin induced these effects while the cell membrane was intact, whereas doxorubicin and daunorubicin led to immediate loss of membrane integrity. Programmed cell death is characterised by preservation of membrane integrity in order to allow removal of apoptotic bodies, whereas cell rupture is an early event in necrosis. We therefore suggest that, in our experimental settings, doxorubicin- and daunorubicin-induced cell death occurs by necrosis, while aclarubicin induces programmed cell death.
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PMID:Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis. 1237 Apr 96

Apoptosis is the mode of cell death in retinitis pigmentosa, a group of retinal degenerative disorders primarily affecting rod photoreceptors. Although caspases have been demonstrated to play a central role in many incidences of apoptosis, accumulating evidence suggests that they may not be required for all forms of apoptotic cell death. The present study examined the mechanism of cell death in two in vivo models of photoreceptor apoptosis: the retinal degeneration (rd) mouse, a naturally occurring mutant model, and N-methyl-N-nitrosourea-induced retinal degeneration. Specifically, we examined the activation status of caspase-9, -8, -7, -3, and -2 and determined the caspase requirements for cytochrome c release, DNA fragmentation, and apoptosis-associated proteolysis of specific caspase substrates. We show that apoptosis in both in vivo models is independent of caspase-9, -8, -7, -3, and -2 activation. DNA fragmentation occurs in the absence of caspase-mediated ICAD (inhibitor of caspase-activated DNase) proteolysis, suggesting that an alternative endonuclease is responsible for DNA cleavage in these models. Importantly, we show that apoptosome activation is prevented because of an absence of mitochondrial cytochrome c release. Experiments performed using a cell-free system indicate that cytochrome c-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochrome c-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. In the rd mouse, however, this age-related downregulation of apoptotic proteins was not observed, highlighting a critical feature of this model and the prevention of cytochrome c release as an apical event in caspase-independent apoptosis in this system.
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PMID:Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration. 1284 76


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