RESUMO
The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Especificidade por Substrato , Proteína bcl-X/metabolismoRESUMO
Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.
Assuntos
Apoptose/fisiologia , Membranas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Motivos de Aminoácidos , Animais , Humanos , Camundongos , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismoRESUMO
In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/fisiologia , Animais , Células Cultivadas , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Multimerização Proteica , Transporte ProteicoRESUMO
The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different 'modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.
Assuntos
Apoptose , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Humanos , Ligação ProteicaRESUMO
During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation.
Assuntos
Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Focalização Isoelétrica , Ponto Isoelétrico , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Tirosina/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Granzimas/farmacologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citocromos c/metabolismo , Células HeLa , Humanos , Células Matadoras Naturais/imunologia , Mitocôndrias/metabolismo , Piperazinas/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.
Assuntos
Apoptose/fisiologia , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismo , Animais , Linhagem Celular Transformada , Citocromos c/genética , Citocromos c/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Apoptose/imunologia , Citotoxicidade Imunológica , Transdução de Sinais/imunologia , Antígenos de Diferenciação de Linfócitos T/toxicidade , Apoptose/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/imunologia , Membranas Intracelulares/metabolismo , Células Jurkat , Células Matadoras Naturais/imunologia , Lipídeos de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaAssuntos
Apoptose/fisiologia , Fracionamento Celular/métodos , Grupo dos Citocromos c/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Animais , Proteínas de Bactérias , Caspases/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Digitonina/farmacologia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Indicadores e Reagentes/farmacologia , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/fisiologia , Modelos Biológicos , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Estreptolisinas/farmacologiaRESUMO
Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Grupo dos Citocromos c/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Ciclosporina/farmacologia , Feminino , Células HL-60 , Células HeLa , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/efeitos da radiação , Membranas Intracelulares/ultraestrutura , Oócitos/fisiologia , Oócitos/ultraestrutura , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologia , Raios Ultravioleta , Xenopus laevis , Proteína X Associada a bcl-2RESUMO
Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.
Assuntos
Apoptose , Grupo dos Citocromos c/metabolismo , Citocromos c , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Cavalos , Lisina/análogos & derivados , Lisina/metabolismo , Metilação , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oócitos , Peptídeo Hidrolases/metabolismo , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.
Assuntos
Apoptose , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Caspases/metabolismo , Sistema Livre de Células , Grupo dos Citocromos c/metabolismo , Epitopos/química , Feminino , Humanos , Células Jurkat , Cinética , Dados de Sequência Molecular , Oócitos/fisiologia , Proteínas/imunologia , Proteínas Recombinantes/metabolismo , Proteínas de Xenopus , Xenopus laevis , Proteína bcl-XRESUMO
Microcinematography was applied to the analysis of the kinetics of apoptotic cell death. Apoptosis was found to be a process that proceeds in different cells at different times after an initial stress, and therefore kinetic studies of apoptotic events in bulk cultures can be problematic. Using single cell analysis we found that stronger apoptotic stimuli induce an earlier onset of apoptosis, but that there is no relationship between time of onset and duration of the apoptotic process. That is, cells that initiate apoptosis shortly after induction do not proceed more rapidly through the process. This suggests an all-or-non-mechanism that is supported by some models of the biochemical pathways of apoptosis.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Tamanho Celular , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Fenótipo , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Raios Ultravioleta/efeitos adversosRESUMO
During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Membranas Intracelulares/fisiologia , Mitocôndrias Hepáticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Adenilato Quinase/metabolismo , Alameticina/farmacologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Sistema Livre de Células , Grupo dos Citocromos c/metabolismo , Citosol/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Cinética , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Oócitos/fisiologia , Peptídeo Hidrolases/metabolismo , Permeabilidade , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Xenopus laevis , Proteína X Associada a bcl-2RESUMO
Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.
Assuntos
Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Animais , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Caspase 10 , Caspase 2 , Caspase 3 , Caspase 6 , Caspase 7 , Caspase 8 , Caspase 9 , Extratos Celulares , Ativação Enzimática , Humanos , Células Jurkat , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , CoelhosRESUMO
In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.
Assuntos
Apoptose/fisiologia , Caspases , Cisteína Endopeptidases/metabolismo , Grupo dos Citocromos c/metabolismo , Lamina Tipo B , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspase 3 , Sistema Livre de Células , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Feminino , Técnicas In Vitro , Laminas , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Óvulo/metabolismo , OxirreduçãoRESUMO
In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
Assuntos
Apoptose , Grupo dos Citocromos c/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Proteínas de Transporte/metabolismo , Extratos Celulares , Sistema Livre de Células , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Potenciais da Membrana , Proteínas dos Microfilamentos/metabolismo , Óvulo , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Proteínas Recombinantes , XenopusRESUMO
Previous studies have established that a relationship exists between apoptosis and cell surface (ecto-) peptidase activity. Thus dose-dependent increases were found both in ectopeptidase activities and in the proportion of cells undergoing apoptosis in HeLa cell monolayers after exposure to UV and other perturbants causing arrest of DNA synthesis (indirectly or directly as a result of DNA damage). The nature of the correlation made no distinction as to whether an increase in peptidase activity was causal of, or consequential to apoptosis, nor whether the increase was a general response by all cells. As a wider approach to understanding the possible role played by ectopeptidases in apoptosis, we report the effect on expression of a known ectopeptidase, aminopeptidase N (CD13), by a myelomonocytic cell line induced to undergo apoptosis. Using THP-1 cultures exposed to low concentrations of ethanol, we used FACS technology to sort for early apoptotic cells that have an increased ability to sequester the vital dye Hoechst 33342 while excluding nonvital dyes. Apoptosis was verified by light, fluorescence, and transmission electron microscopy, and the presence of DNA fragmentation. These early apoptotic cells showed a significant loss in CD13 labeling. Another surface marker, CD33, behaved similarly, whereas CD14 was lost globally, and not just by the apoptotic cells. Peptidase assays confirmed that an aminopeptidase was shed into the bathing media and that this activity was inhibitable both by bestatin and by a CD13 neutralizing monoclonal antibody. In treated cells, there was no evidence for an increase in cell surface protease activity directed toward a highly aliphatic nonapeptide substrate used as a model for TGF-alpha scission from its precursor form. However, other cell surface proteases of different specificity are presumably responsible for the observed shedding of CD13.
Assuntos
Antígenos de Superfície/sangue , Biomarcadores Tumorais/sangue , Antígenos CD13/sangue , Leucemia Mieloide/sangue , Monócitos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Apoptose/fisiologia , Separação Celular , Meios de Cultura , Etanol/farmacologia , Citometria de Fluxo , Humanos , Monócitos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
A moderate sustained rise in intracellular ionised calcium has been observed to be associated with apoptosis occurring in many experimental systems. The application of extracellular and intracellular chelators of calcium has been reported to produce a decrease in apoptosis, while the addition of calcium ionophores often increases apoptosis. These findings, together with the observation of calcium-induced internucleosomal DNA cleavage in isolated nuclei, have suggested that DNA cleavage (and apoptosis) is a calcium-dependent process. However, a number of studies have shown that apoptosis is not always associated with a rise in the level of intracellular ionised calcium. In the present study, calcium chelators were found to induce apoptosis in cultured cells, concomitant with a decrease in both intracellular ionised calcium and total cell calcium content. Decreased intracellular ionised magnesium was also induced by extracellular chelators. These findings provide further evidence that a raised intracellular ionised calcium is not universally present during the induction of apoptosis. It is proposed that loss of calcium homeostasis, rather than a sustained rise in cytosolic calcium, is a determining factor in cell death by apoptosis.