RESUMO
Recent work on the initial switches that trigger cell death has revealed surprising inventions of nature that ensure the ordered suicide of a cell that has been selected for demise. Particularly intriguing is how a signal--the release of cytochrome c from the mitochondria--is translated into the activation of the death cascade, which leads to a point of no return. Now there is new understanding of how this crucial process is delicately handled by a cytosolic signalling platform known as the apoptosome. The formation of the apoptosome and the activation of its effector, caspase-9, reveals a sophisticated mechanism that might be more common than was initially thought.
Assuntos
Apoptose/genética , Apoptossomas/fisiologia , Transdução de Sinais , Animais , Apoptossomas/metabolismo , Caspases Efetoras/metabolismo , Caspases Efetoras/fisiologia , Caspases Iniciadoras/metabolismo , Caspases Iniciadoras/fisiologia , Citosol/metabolismo , Nucleotídeos de Desoxiadenina/fisiologia , Dimerização , Humanos , Mediadores da Inflamação/fisiologia , Proteínas de Membrana/fisiologia , Modelos Biológicos , Modelos MolecularesRESUMO
Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2'-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Centro Germinativo/enzimologia , Centro Germinativo/patologia , Linfopenia/enzimologia , Linfopenia/patologia , Adenosina Desaminase/metabolismo , Animais , Apoptose/genética , Apoptose/imunologia , Líquido Ascítico/citologia , Líquido Ascítico/enzimologia , Líquido Ascítico/imunologia , Líquido Ascítico/patologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/enzimologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Nucleotídeos de Desoxiadenina/biossíntese , Nucleotídeos de Desoxiadenina/fisiologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunoglobulina M/biossíntese , Ativação Linfocitária/genética , Contagem de Linfócitos , Linfopenia/genética , Linfopenia/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/fisiologia , Baço/enzimologia , Baço/imunologia , Baço/metabolismo , Baço/patologiaRESUMO
We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Grupo dos Citocromos c/fisiologia , Precursores Enzimáticos/antagonistas & inibidores , Etoposídeo/farmacologia , Proteínas de Choque Térmico/fisiologia , Caspase 3 , Caspase 9 , Nucleotídeos de Desoxiadenina/fisiologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Células U937RESUMO
Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.
Assuntos
Ribonucleotídeos/metabolismo , Rickettsia prowazekii/fisiologia , Difosfato de Adenosina/fisiologia , Células Cultivadas , Monofosfato de Citidina/fisiologia , Nucleotídeos de Desoxiadenina/fisiologia , Nucleotídeos de Desoxiguanina/fisiologia , Regulação Bacteriana da Expressão Gênica , Hidroxiureia/farmacologia , Ribonucleotídeo Redutases/fisiologiaRESUMO
The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.