RESUMO
Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasegamma during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNasegamma functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasegamma expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNasegamma. Our results suggest that DNasegamma provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.
Assuntos
Apoptose , Linfócitos B/citologia , Linfócitos B/enzimologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Perfilação da Expressão Gênica , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos B/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas Mutantes/metabolismo , Necrose , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
In this report, we describe the molecular cloning and characterization of DLAD, a novel mammalian deoxy-ribonuclease homologous to DNase II. The full length cDNA for mouse DLAD has been cloned by polymerase chain reaction. The cDNA contains a 1065 bp open reading frame (ORF) encoding a 354 amino acid protein with a calculated molecular mass of 40 767. The predicted protein for DLAD shares 34.4% identity with DNase II. DLAD is also homologous to three predicted proteins, C07B5.5, F09G8.2 and K04H4.6, from the nematode Caenorhabditis elegans. Furthermore, the third ORF of the fowlpox virus genome is found to encode a DLAD homologue showing 37. 1% identity at the amino acid level. Northern blot analysis reveals that expression of the DLAD mRNA is highly restricted to the liver. DLAD mainly exists as a cytoplasmic protein with divalent cation-independent endonuclease activity and cleaves DNA to produce 3'-phosphoryl/5'-hydroxyl ends. It is active under a wide range of pH with maximum activity at pH 5.2. Among known DNase inhibitors tested, aurintricarboxylic acid and Zn(2+)are found to be effective inhibitors of the DLAD activity.
Assuntos
Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cátions Bivalentes/metabolismo , Clonagem Molecular , DNA/metabolismo , DNA Complementar/química , DNA Complementar/isolamento & purificação , Endodesoxirribonucleases/química , Etiquetas de Sequências Expressas , Células HeLa , Humanos , Hidrólise , Camundongos , Dados de Sequência MolecularRESUMO
In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.
Assuntos
Apoptose , Diferenciação Celular , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Animais , Apoptose/efeitos dos fármacos , Catálise , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , Camundongos , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Ratos , Estaurosporina/farmacologia , Transcrição GênicaRESUMO
The effects of 3-aminobenzamide (3ABm) and benzamide (BAm), known specific inhibitors of poly(ADP-ribose) polymerase (PARP), on actinomycin D (Act D)-induced apoptosis in HL-60 cells were examined. These inhibitors had no appreciable effect on apoptotic DNA fragmentation, chromatin condensation or PARP restriction cleavage, but clearly inhibited morphological changes, especially nuclear fragmentation and apoptotic-body formation, in a dose-dependent manner. These results suggest that the synthesis of ADP-ribose polymers is not essential for the progression of apoptotic DNA fragmentation and chromatin condensation, but is required in the processes leading to nuclear fragmentation and the subsequent apoptotic-body formation during apoptosis in HL-60 cells.
Assuntos
Apoptose/efeitos dos fármacos , Núcleo Celular/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/fisiologia , Aminobenzoatos/farmacologia , Benzamidas/farmacologia , Benzoatos/farmacologia , Ácido Benzoico , Western Blotting , Núcleo Celular/efeitos dos fármacos , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/análise , meta-AminobenzoatosRESUMO
U1 cells, a subclone of U937 cells chronically infected with human immunodeficiency virus type 1 (HIV-1), produced HIV-1 only in the presence of inducers such as 12-O-tetradecanoxylphorbol 13-acetate (TPA) or tumor necrosis factor (TNF)-alpha. The expression of HIV-antigen on U1 cells induced by TPA or TNF-alpha was found to be prevented by sodium 5,6-benzylidene-L-ascorbate (SBA) in a concentration-dependent manner. Treatment of U1 cells with SBA in the presence of inducers resulted in cell death with cell shrinkage, chromatin condensation and DNA fragmentation into nucleosomal oligomers, characteristics of apoptosis. In contrast, SBA had scarcely any apoptotic effect on U1 cells in the absence of inducers. SBA did not also induce apoptosis in parental U937 cells in the presence or absence of inducers. These results suggest that HIV-replicating U1 cells selectively undergo apoptosis on treatment with SBA.
Assuntos
Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Ácido Ascórbico/análogos & derivados , Compostos de Benzilideno/farmacologia , HIV-1/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Antígenos HIV/análise , Humanos , Estrutura Molecular , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Although apoptotic cell death has been suggested to be involved in ischemic injury of the brain, the precise mechanisms of ischemic neuronal cell death are unknown. Here, we examined the biochemical feature of apoptosis (i.e. DNA fragmentation) in male spontaneously hypertensive rats (5-7 months old) subjected to photothrombotic distal middle cerebral artery (MCA) occlusion. After MCA occlusion, the brain was cut in a cryostat to produce a standard coronal block and samples were dissected from the regions corresponding to the ischemic core, penumbra and contralateral control areas. Changes in cerebral blood flow (CBF) were monitored at 1 mm posterior and 2-4 mm lateral to the bregma by means of a laser-Doppler flowmetry. After MCA occlusion, CBF was decreased to 72+/-18 (+/-S.D.), 50+/-14, and 35+/-11% of the control values at 2, 3, and 4 mm from the midline, respectively. DNA fragmentation characteristics of apoptosis were examined in these samples by conventional and pulse-field gel electrophoresis. On the conventional gel electrophoresis, nucleosomal DNA fragmentation was detected in the penumbral zone at 6 h after MCA occlusion. Large DNA fragments of 50 and 20 kbp were detected in the penumbral zone and also in the ischemic core region at 3 h after distal MCA occlusion. The large DNA fragments seen on the pulse-field gel elecrophoresis were further degraded to small DNA fragments at 6 h after MCA occlusion in the penumbral zone but not in the core regions. The evolving DNA fragmentation was observed between 3 and 6 h after the onset of brain ischemia in the penumbra, suggesting that apoptosis may contribute to the development of ischemic infarction.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Fragmentação do DNA , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Animais , Circulação Cerebrovascular , Eletroforese em Gel de Campo Pulsado , Masculino , Neurônios/patologia , Ratos , Ratos Endogâmicos SHRRESUMO
High-performance liquid chromatography revealed that sodium 5,6-benzylidene-L-ascorbate (SBA), dissolved in distilled water, was gradually decomposed into ascorbic acid and benzaldehyde. Among these three compounds, ascorbic acid showed the most potent cytotoxic activity. The cytotoxic activity of each compound was significantly reduced during degradation in culture medium. Agarose gel electrophoresis and fluorometric determination of DNA revealed that ascorbic acid, as well as SBA, induced DNA fragmentation into nucleosomal oligomers in human myelogenous leukemic cell lines, but not in freshly isolated human peripheral blood cells. The results suggest that antitumor activity of SBA might be at least in part mediated by the action of ascorbic acid, a degradation product of SBA.
Assuntos
Antineoplásicos/farmacologia , Ácido Ascórbico/análogos & derivados , Compostos de Benzilideno/farmacologia , DNA/metabolismo , Animais , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Benzaldeídos/metabolismo , Compostos de Benzilideno/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , CamundongosRESUMO
We report here the cDNA cloning and functional analysis of Xenopus DNase gamma (xDNase gamma). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase gamma protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase gamma mRNA is expressed in spleen, liver, testis, and ovary. xDNase gamma expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase gamma results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase gamma in apoptosis during amphibian metamorphosis.
Assuntos
Apoptose , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Endodesoxirribonucleases/química , Larva/anatomia & histologia , Larva/crescimento & desenvolvimento , Metamorfose Biológica , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/química , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
We describe here the characterization of the so far identified human DNase I family DNases, DNase I, DNase X, DNase gamma, and DNAS1L2. The DNase I family genes are found to be expressed with different tissue specificities and suggested to play unique physiological roles. All the recombinant DNases are shown to be Ca(2+)/Mg(2+)-dependent endonucleases and catalyze DNA hydrolysis to produce 3'-OH/5'-P ends. High activities for DNase I, DNase X, and DNase gamma are observed under neutral conditions, whereas DNAS1L2 shows its maximum activity at acidic pH. These enzymes have also some other peculiarities: different sensitivities to G-actin, aurintricarboxylic acid, and metal ions are observed. Using a transient expression system in HeLa S3 cells, the possible involvement of the DNases in apoptosis was examined. The ectopic expression of each DNase has no toxic effect on the host cells; however, extensive DNA fragmentation is observed only in DNase gamma-transfected cells after the induction of apoptosis. Furthermore, DNase gamma is revealed to be located at the perinuclear region in living cells, and to translocate into the nucleus during apoptosis. Our results demonstrate that DNase I, DNase X, DNase gamma, and DNAS1L2 have similar but unique endonuclease activities, and that among DNase I family DNases, DNase gamma is capable of producing apoptotic DNA fragmentation in mammalian cells.
Assuntos
Apoptose , Desoxirribonuclease I/química , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Família Multigênica , Actinas/farmacologia , Transporte Ativo do Núcleo Celular/genética , Adulto , Sequência de Aminoácidos , Apoptose/genética , Linhagem Celular , Desoxirribonuclease I/antagonistas & inibidores , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/biossíntese , Endodesoxirribonucleases/genética , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Cromossomo X/enzimologiaRESUMO
Three distinct endonucleases (DNase alpha, beta and gamma) were isolated from rat thymocyte nuclei. These three DNases differed in chromatographic behaviors and in apparent molecular masses. The cognate form of acidic DNases alpha and beta did not require divalent cations for their activities, whereas DNase gamma was a neutral endonuclease that required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. These enzymes digested HeLa S3 cell nuclear chromatin to the nucleosomal fragments characteristic of apoptosis. Interestingly, apoptotic rat thymocyte nuclei induced by gamma-ray irradiation or glucocorticoid retained considerable activity of DNase gamma, whereas their activities of DNases alpha and beta were markedly reduced. These results indicate that in rat thymocytes DNase gamma is involved in DNA fragmentation during apoptosis.
Assuntos
Núcleo Celular/enzimologia , Desoxirribonucleases/isolamento & purificação , Linfócitos/enzimologia , Timo/ultraestrutura , Animais , Apoptose , Cálcio/farmacologia , Cátions Bivalentes , Cromatina/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Masculino , Peso Molecular , Ratos , Ratos Wistar , Timo/enzimologia , Zinco/farmacologiaRESUMO
DNase II is a well-known deoxyribonuclease (DNase) that catalyzes the hydrolysis of DNA into oligonucleotides under acidic conditions. We have identified a novel DNase that shows homology to DNase II, named DLAD, from a search of an expressed sequence tag data base. The full-length cDNA for rat DLAD cloned by polymerase chain reaction encodes a 356-amino acid polypeptide containing a putative N-terminal signal peptide and 5 potential N-glycosylation sites; there is a predicted catalytic domain resemblance to rat DNase II. The predicted DLAD translation product shares 32.9% identity with DNase II. Interestingly, expression of the DRAD mRNA is highly restricted to the liver. A Myc-His tagged recombinant DLAD recovered mainly from the cytoplasm of transfected HeLa S3 cells has a divalent cation-independent DNase activity. The DLAD activity prefers acidic conditions to neutral. The recombinant protein expressed in HeLa S3 cells inhibits the expression of GFP- and lac Z-expression vectors, suggesting that DLAD may play a role in elimination of exogenous DNA. Identification of the full-length cDNA for DLAD would lead to an understanding of the physiology of this DNase II-like molecule.
Assuntos
DNA Complementar/genética , Desoxirribonucleases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Endodesoxirribonucleases/genética , Etiquetas de Sequências Expressas , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Óperon Lac , Proteínas Luminescentes/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
We report the molecular cloning of cDNAs encoding porcine and human DNase II and the genomic structure of the human DNase II gene. The full length cDNAs for porcine and human DNase II were isolated by polymerase chain reaction on the basis of amino acid sequences determined for the tryptic peptides of porcine liver DNase II. The porcine and human cDNAs contain 1095 and 1083 bp open reading frames, respectively, and encode 364 and 360 amino acid proteins with calculated molecular masses of 40,157 and 39,555, respectively. The amino acid sequencing of purified porcine DNase II reveals two N-termini with corresponding sequences present within the same open reading frame, suggesting proteolytic processing for the covalently bonded subunit structure of DNase II. Northern blot analysis demonstrated that a single transcript of 2.0 kb mRNA coding for DNase II is ubiquitously expressed in human tissues. A database search revealed that the human genomic sequence of chromosome 19p13.2 contains the DNase II gene. Characterization of the genomic sequence showed that the DNase II gene consists of six exons separated by five introns whose splice acceptor/donor sites agree with the GT/AG rule.
Assuntos
Endodesoxirribonucleases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Humanos Par 19/genética , Clonagem Molecular , Éxons/genética , Humanos , Íntrons/genética , Fígado/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional/fisiologia , Splicing de RNA/genética , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SuínosRESUMO
An endonuclease named DNase gamma has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675-681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase gamma. A 1.6 kb mRNA coding for the DNase gamma precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase gamma mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase gamma reveals that full-length DNase gamma, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase gamma, from which the N-terminal precursor has been removed, has the same properties as purified DNase gamma: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase gamma cDNA, exogenously introduced DNase gamma is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase gamma in DNA fragmentation and nuclear structural changes during apoptosis.
Assuntos
Apoptose/genética , DNA Complementar/genética , Endodesoxirribonucleases/genética , Animais , Apoptose/fisiologia , Sequência de Bases , Cromatina/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Complementar/biossíntese , DNA Complementar/química , DNA Complementar/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Ativação Enzimática , Glutationa Transferase/biossíntese , Células HeLa , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We previously found that a novel DNA endonuclease named DLAD (DNase II-Like Acid DNase) is specifically expressed in murine liver. Here, we describe the isolation and characterization of the human DLAD and mouse Dlad genes. Both DLAD and Dlad consist of 6 exons. DLAD encodes a 361 amino acid protein sharing 34.6% amino acid identity with human DNase II. Although a recombinant protein for the putative human DLAD has a divalent cation-independent acid DNase activity, expression of the DLAD mRNA containing the entire open reading frame was not detected in any human tissues tested, except for lung, in which a short 1.1 kb transcript lacking the first two exons is expressed. Interestingly, sequence analysis of Dlad showed that the 1st exon of the urate oxidase gene, Uox, is located on the opposite strand in its 5'-flanking region. The head-to-head organization of DLAD and UOX is conserved in the human genomic sequence. Promoter analysis revealed that the intergenic region between Dlad and Uox has promoter activity for both the Dlad and Uox directions, however, the corresponding human genomic fragment has promoter activity only for DLAD. It is known that murine Uox is expressed only in the liver, whereas human UOX has been inactivated as a pseudogene. On the basis of these results, the expression of DLAD/Dlad and UOX/Uox is suggested to be coordinated by a common regulatory mechanism(s), and the balance between the two enzymes is thought to be important for maintaining the purine nucleotide pool in the liver.
Assuntos
Endodesoxirribonucleases/genética , Homologia de Genes , Urato Oxidase/genética , Região 5'-Flanqueadora , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Endodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ratos , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.
Assuntos
Endodesoxirribonucleases/química , Baço/enzimologia , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Cromatografia Líquida de Alta Pressão , Desoxirribonuclease I/química , Desoxirribonuclease I/classificação , Endodesoxirribonucleases/classificação , Endodesoxirribonucleases/efeitos dos fármacos , Endodesoxirribonucleases/metabolismo , Magnésio/farmacologia , Masculino , Dados de Sequência Molecular , Ratos , Ratos Wistar , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
We report here on the nucleotide sequence of the cDNA encoding human DNase gamma, which is a candidate for an apoptotic endonuclease. The cDNA clone isolated from a human spleen cDNA library is composed of a 918 bp open reading frame encoding a 305 amino acid precursor protein for DNase gamma. Northern blot analysis reveals that the expression of a single transcript of 1.5 kb DNase gamma mRNA is detected in the spleen and liver. The chromosomal localization of DNase gamma gene is mapped to chromosome 3 at region p21.1-p14.2 by fluorescence in situ hybridization (FISH). Characterization of thioredoxin-DNase gamma fusion protein (Trx-hDNase gamma) shows that the recombinant protein has a Ca(2+)/Mg(2+)- or Mn(2+)-dependent endonuclease activity that cleaves chromatin DNA to nucleosomal units. The optimum pH is around 7.2. Zn(2+) and aurintricarboxylic acid (ATA) inhibits the activity in dose-dependent manners. These properties are identical to those of purified DNase gamma.
RESUMO
We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase gamma activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo (in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase gamma. DNase gamma activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
Assuntos
Apoptose , Endodesoxirribonucleases/isolamento & purificação , Endodesoxirribonucleases/metabolismo , Linfócitos T/enzimologia , Linfócitos T/patologia , Animais , Masculino , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
Analyses of cleavage ends of DNA fragments in apoptotic rat thymocytes induced by gamma-ray irradiation or by treatment with dexamethasone revealed that in both cases the fragments produced had 3'-hydroxyl (OH) and 5'-phosphoryl (P) ends of DNA chains. Rat thymocyte nuclei contained at least three endonuclease activities (deoxyribonucleases alpha, beta and gamma) that were able to cleave chromatin to mononucleosomal and oligonucleosomal fragments. The nuclei of apoptotic rat thymocytes induced by gamma-ray irradiation or dexamethasone retained considerable deoxyribonuclease gamma activity, but not alpha or beta deoxyribonuclease activity. During the induction of apoptosis, treatment with cycloheximide, which suppressed apoptosis, resulted in marked decreases of deoxyribonucleases alpha and beta activities. After release of cycloheximide inhibition, DNA fragmentation associated with apoptosis occurred in the cycloheximide-treated thymocyte nuclei, in which deoxyribonuclease gamma activity was only observed. The purified deoxyribonucleases alpha and beta were divalent cation-independent acidic endonucleases, which were separated on a CM5PW column by HPLC. The molecular masses of deoxyribonucleases alpha and beta were 28 kDa and 30 kDa, respectively, as determined by TSK G-2000SW gel-filtration HPLC, and both were 32 kDa in molecular mass as determined by SDS/PAGE. In contrast, deoxyribonuclease gamma, a neutral endonuclease, required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. The molecular mass of deoxyribonuclease gamma was 31 kDa and 33 kDa when measured by gel filtration and SDS/PAGE, respectively. Under these optimal conditions, deoxyribonuclease gamma was shown to produce 3'-OH/5'-P ends of nucleosomal DNA fragments, while deoxyribonucleases alpha and beta both formed DNA fragments with 3'-P/5'-OH ends. The ends formed by cleavage with deoxyribonuclease gamma were the same as those produced in apoptotic rat thymocytes. On the basis of these results, it seems likely that deoxyribonuclease gamma is responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
Assuntos
Apoptose , Endonucleases/fisiologia , Linfócitos T/enzimologia , Animais , Cátions Bivalentes , Núcleo Celular/enzimologia , Endonucleases/antagonistas & inibidores , Endonucleases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Masculino , Peso Molecular , Ratos , Ratos Wistar , Linfócitos T/citologia , Timo/citologiaRESUMO
DNase gamma has been suggested to be an endonuclease responsible for thymic apoptosis in mammals. Using the frog, Xenopus laevis, we examined whether any hematopoietic cells other than thymocytes have DNase gamma activity. The activity gel assays and HeLa cell nuclear assays for DNase revealed that nuclei from red blood cells and cells in liver (an erythropoietic organ) contain DNase gamma-like activities (molecular mass of 38 and 36 kDa) indicated by their biochemical features (internucleosomal DNA cleavage, requirement of Ca2+ and Mg2+, and sensitivity to Zn2+). Distribution of these two (38 and 36 kDa) forms of enzyme was determined by cell fractionation analysis of liver cells: the 38 kDa form of enzyme was found in erythrocytes/erythroblasts and hepatocytes, whereas the 36 kDa form was in lymphocytes/macrophages. Interestingly, these two enzyme activities increased during metamorphic climax, at which time larval-type cells are converted to adult ones. It thus appears that these gamma-type DNases are involved in programmed cell death during metamorphosis.