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1.
J Biol Chem ; 276(30): 28309-13, 2001 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-11390368

RESUMO

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.


Assuntos
Caspase 1/metabolismo , Proteínas/química , Proteínas/fisiologia , Sequência de Aminoácidos , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Células Cultivadas , DNA Complementar/metabolismo , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Microscopia Confocal , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas
2.
Biochim Biophys Acta ; 1451(1): 59-72, 1999 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-10446388

RESUMO

Induction of the antiproliferative and antiviral state by IFNs (type I and II) is dramatically impaired in HepG2 cells. We show here that RNase L, IDO, GBP-2 and iNOS genes normally expressed as a secondary response to IFN are no longer inducible in HepG2 cells, while induction of primary response genes (IRF-1, PKR, p48-ISGF3gamma, 2-5AS, 6-16 and p56-(trp)tRNA) are unaffected. On the basis of previous data implicating transcription factor IRF-1 in the induction of some IFN-induced genes, we tested the effects of transfecting an IRF-1 oligonucleotide antisense in HeLa cells and found specifically impaired IFN induction of secondary response genes (RNase L, IDO and GBP-2). This raised the possibility that IRF-1 was defective in HepG2 cells. However, some molecular and biochemical analyses reveal that IRF-1 is induced normally by IFNs and retains its normal size, cellular location, phosphorylation status and ability to bind the IDO promoter in vitro. Therefore, we conclude that although the primary response pathway is fully functional, some aspects of the secondary pathway involving IRF-1 (but not IRF-1 itself) are defective in HepG2 cells. It may be possible that the promoter region of these deficient HepG2-genes requires an unidentified transcription factor in addition to de novo IRF-1, which could be elicited by a cooperative activator.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação ao GTP , Regulação da Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , Fosfoproteínas/genética , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Vírus da Encefalomiocardite/efeitos dos fármacos , Endorribonucleases/biossíntese , Imunofluorescência , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase , Fator Regulador 1 de Interferon , Camundongos , Oligonucleotídeos Antissenso/genética , Fosfoproteínas/biossíntese , Ligação Proteica , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos dos fármacos , Transfecção , Triptofano Oxigenase/biossíntese , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
J Interferon Cytokine Res ; 18(6): 361-8, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9660242

RESUMO

RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involved in the molecular mechanism of interferon (IFN) action. Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regulation of RNA stability in IFN-treated or virus-infected mammalian cells. Knowledge of RNase location within cells may provide additional information about its function. Previous work located RNase as a detergent-soluble molecule in nuclei and cytoplasm. In this study, we demonstrate that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton. A cellular fractionation procedure was used to prepare the cytoskeleton, which was shown to contain 2-5A binding activity not due to cytoplasmic contaminants. In contrast to the cytoplasmic fraction, which contained RNase L with a 2-5A-accessible site, the insoluble RNase molecular form of the cytoskeleton could not be assayed by the classic radiobinding method or the covalent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2-5A site. The 2-5A binding site present in the cytoskeleton was completely masked and not directly accessible to its 2-5A activator. This particular molecular form of RNase can be detected after a specific denaturing-renaturing treatment of the cytoskeleton, which separates the RNase from cytoskeletal proteins, unmasking the 2-5A site. The cytoskeletal RNase was no longer present at this site when cells were stimulated for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data suggest the existence of a pathway that targets the RNase to another subcellular location. To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein kinase C (PKC) to catalyze significant phosphorylation of the RNase.


Assuntos
Citoesqueleto/enzimologia , Endorribonucleases/análise , Nucleotídeos de Adenina/farmacologia , Animais , Membrana Celular/enzimologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Interferons/metabolismo , Camundongos , Oligorribonucleotídeos/farmacologia , Fosforilação , Proteína Quinase C/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
4.
Biochim Biophys Acta ; 1402(2): 139-50, 1998 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-9561800

RESUMO

2',5'-adenylate oligonucleotide (2-5A)-dependent RNase and 2-5A-synthetase are two enzymes of the 2-5A system strongly implicated in the basal control of RNA decay of both interferon-treated and untreated cells. RNase is activated by a 2-5A produced by 2-5A-synthetase, both enzymes being overexpressed by type I-interferon (alpha/beta). We described here for the first time a cell line completely deficient in RNase and its mRNA, while p69 2-5A-synthetase was normally interferon alpha/beta-induced. The complete absence of this RNase in human hepatoma cells (HepG2) was shown using three different methods based on the binding of a [32P]-labeled 2-5A probe of high specific activity to its binding site. Negative Western blotting assay with a specific monoclonal antibody correlated the previous findings. RNase-specific mRNA was not detectable even after treatment of cells with 1000 units/ml of interferon alpha/beta. This is not due to a mutation of the gene because an intronless genomic DNA sequence encoding 2-5A-binding site was cloned and expressed. It is likely that the expression of 2-5A-dependent RNase was impaired at the transcriptional level while having the known IFN alpha/beta-transcriptional regulatory factors as revealed by induction of p69 2-5A-synthetase gene. This may account for a differential activation of 2-5A-dependent RNase and 2-5A-synthetase genes by type I-interferon, and suggests that other members of regulatory transcription factors, different from IRF-1 and STAT proteins, may participate in two different interferon alpha/beta signaling pathways.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Carcinoma Hepatocelular/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Interferon-alfa/farmacologia , Proteínas Repressoras , 2',5'-Oligoadenilato Sintetase/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/genética , Sítios de Ligação , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Interferon beta/farmacologia , Fosfoproteínas/biossíntese , Fosfoproteínas/efeitos dos fármacos , RNA Mensageiro , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas
5.
J Interferon Cytokine Res ; 17(7): 425-9, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9243376

RESUMO

Labeling of unstimulated human Daudi B lymphoblastoid cells with exogenously added [3H]myristate resulted in acylation of a broad spectrum of different proteins, most of which are currently unknown. Among this array of labeled proteins, a unique 74-kDa acylated protein was induced in interferon (IFN)-treated cells. In the present study, we defined the myristylation kinetics of this protein and examined the subcellular distribution before and after activation with IFN-alpha/beta. This acylated protein was detected only at a very low level in the membrane fraction of untreated cells, and its level increased 3-4-fold by treatment with IFN. This induction occurred over a short period of time and was IFN-alpha/beta dose-dependent. No significant induction was observed with IFN-gamma. Incorporation of [3H]myristate was completely abolished by cycloheximide. The fatty acid associated with this protein was probably linked to a nascent chain through an amide linkage, as it was not released by alkaline hydroxylamine treatment and was identified as myristic acid by HPLC after its release from the polypeptide chain by acid methanolysis. In contrast to other IFN-induced proteins, whose synthesis started at 10 h and was maintained for 20 h, this protein was present in the plasma membrane for a short period of time, between 4 and 6 h after IFN-alpha/beta treatment, and was no longer present in this cellular compartment. This event appears to be transient and suggests that a degradation or a negative regulation of transcription starts from 6-7 h after continuous IFN treatment. As many other myristylated proteins are implicated in cellular regulation, it is possible that this 74-kDa protein may have a regulatory role in cell proliferation and the inhibition of viral replication.


Assuntos
Interferon Tipo I/farmacologia , Ácidos Mirísticos/metabolismo , Proteínas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Humanos , Cinética , Peso Molecular , Ácido Mirístico , Ligação Proteica , Regulação para Cima
6.
Artigo em Francês | MEDLINE | ID: mdl-1838756

RESUMO

Four hundred and seventy height Culdocenteses were carried out in cases of possible ectopic pregnancy between the 20th September 1986 and 31st December 1987. Culdocentesis was considered to be positive if 2 cm3 or more of dark non-coagulated blood was removed, and negative if only a yellow liquid or blood stained serum was removed. It was not conclusive if nothing could be aspirated or if the blood was coagulated. Of the 94 cases where culdocentesis was positive, 74 were found to have an ectopic pregnancy. There were 20 false positive cases (due to 5 haemorrhagic ruptures of follicles, 3 refluxes of menstrual blood, 2 with other aetiology, and 10 without known cause). There were 21 cases of ectopic pregnancy in the 293 cases where culdocentesis was non-conclusive. Of the 91 cases where culdocentesis was negative, a second culdocentesis showed an ectopic pregnancy. It was positive 11 days after the first. In our series this diagnostic test was reliable in 77.1% of cases. Laparotomy was carried out in 22.3% of cases and only 18.6% had to have laparoscopy thanks to the use of culdocentesis.


Assuntos
Culdoscopia/normas , Inalação , Gravidez Ectópica/diagnóstico , Protocolos Clínicos/normas , Culdoscopia/métodos , Árvores de Decisões , Estudos de Avaliação como Assunto , Feminino , Humanos , Laparoscopia , Laparotomia , Gravidez , Gravidez Ectópica/epidemiologia , Gravidez Ectópica/cirurgia , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tunísia/epidemiologia , Ultrassonografia Pré-Natal
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