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)

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.
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PMID:Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease. 1646 18

Curcumin is a natural pigment that has been shown to induce cell death in many cancer cells; however, the death mode depends on the cell type and curcumin concentration. Here we show that, in Jurkat cells, 50 micromol/L curcumin severely lowers cell survival and induces initial stage of chromatin condensation. It also induces caspase-3, which is sufficient to cleave DNA fragmentation factor 45 [DFF45/inhibitor of caspase-activated DNase (ICAD)], the inhibitor of DFF40/CAD endonuclease. However, the release of DFF40/CAD from its inhibitor does not lead to oligonucleosomal DNA degradation in curcumin-treated cells. Moreover, curcumin treatment protects cells from UVC-induced oligonucleosomal DNA degradation. In biochemical experiments using recombinant DFF activated with caspase-3, we show that curcumin inhibits plasmid DNA and chromatin degradation although it does not prevent activation of DFF40/CAD endonuclease after its release from the inhibitor. Using DNA-binding assay, we show that curcumin does not disrupt the DNA-DFF40/CAD interaction. Instead, molecular modeling indicates that the inhibitory effect of curcumin on DFF40/CAD activity results from curcumin binding to the active center of DFF40/CAD endonuclease.
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PMID:Curcumin induces caspase-3-dependent apoptotic pathway but inhibits DNA fragmentation factor 40/caspase-activated DNase endonuclease in human Jurkat cells. 1664 63

Apoptotic endonuclease is a key enzyme that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals such as the Fas ligand, ionizing radiation, and anticancer agents. An endonuclease that is activated specifically by caspase-3 has been identified in humans and mice. The human gene for this protein has been termed DFF40 (DNA fragmentation factor, 40-kd subunit) or caspase-activated nuclease (CPAN), whereas the mouse homologue has been named caspase-activated deoxyribonuclease (CAD). Although CAD/DFF40 is known as a major apoptotic nuclease, mice lacking inhibitor of CAD (ICAD) (also known as DFF45) are viable and still show DNA fragmentation, suggesting that alternative endonucleases play an important role during apoptosis. Endonuclease G has been reported to possibly be responsible for DNA fragmentation in various cells during apoptosis. Furthermore, we also have found that apurinic/apyrimidinic endonuclease 1 (Ape1) and its N-terminal-truncated form (AN34) are involved in DNA fragmentation during apoptosis in leukemia cells. In this review, we describe the features of several endonucleases that are involved in the apoptosis of human leukemia cells. Apoptotic endonuclease may vary among different leukemia cell types.
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PMID:Endonuclease activation and chromosomal DNA fragmentation during apoptosis in leukemia cells. 1686 99

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.
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PMID:Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells. 1702 93

Mildly affected individuals from xeroderma pigmentosum complementation group G (XP-G) possess single amino acid substitutions in the XPG protein that adversely affects its 3' endonuclease function in nucleotide excision repair. More serious mutations in the XPG gene generate truncated or unstable XPG proteins and result in a particularly early and severe form of the combined XP/CS complex. Following UV irradiation, cells from such XP-G/CS patients enter apoptosis more readily than other DNA repair-deficient cells. Here, we explore the mechanisms by which UV triggers the apoptotic cell death program in XP-G and XP-G/CS primary fibroblasts. Activation of the CD95 signalling pathway occurs within minutes and it is the earliest detectable post-UV event in such cells. This is rapidly followed by activation of caspase-8 then caspase-3. Several hours later caspase-9 becomes activated and the mitochondrial membrane potential drops, but without any obvious prior release of cytochrome c. Although p53 accumulates in XPG-deficient cells after UV irradiation, use of RNA interference demonstrates that p53 is not required for their UV-induced apoptotic response. p53 ablation of wild-type fibroblasts reduces MDM2 mRNA levels, inhibits accumulation of the 90kDa/92kDa Mdm2 isoforms, and prevents the nuclear relocalisation of Mdm2 after UV treatment. The same post-UV effects occur in XPG-deficient cells that express normal p53 levels. These results emphasise the importance of the extrinsic apoptotic pathway and aberrant Mdm2 events for the severe UV-induced apoptosis of XPG-deficient primary fibroblasts. XP-G/CS cells constitutively overexpress the pro-apoptotic Bax protein and a long isoform of the E2F1 transcription factor that controls S phase entry, which may prime them to enter apoptosis very readily after UV irradiation.
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PMID:UV-induced apoptosis in XPG-deficient fibroblasts involves activation of CD95 and caspases but not p53. 1720 56

Neurons are targets of toxicity induced by the human immunodeficiency virus (HIV)-1 protein Tat (transactivator of transcription). Exposure to Tat increases [Ca(2+)](i) in striatal neurons and activates multiple cell death pathways. In earlier studies the authors showed that Tat activated both caspase-3 and endonuclease-G, a caspase-independent effector of apoptosis, and that Tat-induced neurotoxicity was not attenuated by a caspase-3 inhibitor. Because Tat activates multiple, parallel death pathways, the authors attempted to reduce Tat-induced neurotoxicity by manipulating signaling pathways upstream of mitochondrial apoptotic events. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a negative regulator of Akt/PKB (protein kinase B) phosphorylation, was chosen as a target for silencing. Akt/PKB activity directs multiple downstream pathways mediated by GSK3beta, BAD, forkhead transcription factors, nuclear factor kappa B (NFkappaB), and others, in a manner that promotes proliferation and survival. Striatal neurons were nucleofected with short interfering RNA (siRNA) vectors targeting PTEN, or a negative-control siRNA. Although Tat(1-86) significantly increased the death of neurons transfected with control construct by 72 h, PTEN-silenced neurons were completely protected. These findings indicate that Akt is a critical intermediary in the direct neurotoxicity induced by HIV-1 Tat, and identify Akt regulation as a possible therapeutic strategy for Tat-induced neurotoxicity in HIV encephalitis (HIVE).
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PMID:Silencing the PTEN gene is protective against neuronal death induced by human immunodeficiency virus type 1 Tat. 1750 78

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process.
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PMID:GP7 induces internucleosomal DNA fragmentation independent of caspase activation and DNA fragmentation factor in NB4 cells. 1754 79

We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5.
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PMID:Engineered apoptotic nucleases for chromatin research. 1762 49

We have found novel functions of scaffold attachment factor-B1 (SAFB) during apoptosis. The experiments showed that SAFB moved into the nucleolus 15 min after the induction of apoptosis and before the release of cytochrome c into the cytoplasm. Two hours later SAFB formed a peri-nucleolar ring-like structure and this occurred after cytochrome c release and before PARP cleavage. Digestion with RNase suggested that the peri-nucleolar ring structure was dependent on RNA integrity and a RNA moiety formed part of this structure. Studies using SAFB deletion mutants showed that the formation of the peri-nucleolar structure was not mediated by the DNA binding (SAP) or the RNA binding (RRM) domain of SAFB but was instead dependent on the S/K and R/E coiled-coil regions: a result suggesting that the structure is formed via protein interactions. In addition, SAFB cleavage was shown to be mediated by caspase-3 and occurred after the formation of the peri-nucleolar ring and after cleavage of PARP (characteristic of proteins having a direct role in apoptosis). A determinant for this cleavage is located in the DNA binding domain and we hypothesize that SAFB may direct the reorganization and segregation of nuclear RNA and DNA prior to endonuclease-mediated DNA cleavage.
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PMID:SAFB re-distribution marks steps of the apoptotic process. 1764 27

Renal cell apoptosis is important in both physiological conditions such as normal renal development and pathological processes affecting the glomerular, vascular or tubulointerstitial compartments. Apoptosis may result in the detrimental loss of cells following many renal diseases or damaging changes, with significant loss of function. In contrast, apoptosis may control and limit inflammatory processes in both the acute and chronic phases of renal disease. Investigators interested in the presence of apoptotic cells in different forms of renal disease and development need methods to accurately determine the level of apoptosis within the kidney. Apoptosis is a gene-driven mode of cell death that may be identified by distinct morphological features, endonuclease-initiated DNA degradation, and by the involvement of specific apoptosis-regulating proteins. Many research papers that analyse the presence of apoptosis use the in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay that detects DNA strand breaks in situ in tissue sections. Localization of activated caspase-3 is now seen as an alternative to TUNEL. This review will discuss some methods of identifying apoptosis in the kidney, using both morphological and biochemical or molecular characteristics, and also discuss some of the pitfalls of entire reliance on biochemical means of apoptotic cell identification without some morphological checks and balances. Although there are some caveats to the methods for identifying apoptotic cells in renal disease, those investigators who take the time to undertake such analysis often gain insightful data that provide explanations for the disease or condition being studied.
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PMID:Identification and quantification of apoptosis in the kidney using morphology, biochemical and molecular markers. 1780 68


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