RESUMEN
Dynamic protein-protein interactions between proapoptotic and pro-survival Bcl-2 family members regulate outer-mitochondrial membrane permeabilization and cytochrome c release, key events in the path to apoptosis. Their relative levels often dictate the fate of a cell following an apoptotic stimulus. However, in cancer cells, the pro-survival Bcl-2 family members are frequently upregulated, thereby creating a constitutive block to apoptosis and resulting in continued cell survival under conditions that normally result in cell death. Because many chemotherapeutics used to treat cancer also trigger apoptosis, this upregulation of pro-survival members also contributes to resistance to conventional cancer therapies. Strategies that inactivate pro-survival Bcl-2 family members therefore suggest a means by which this downstream block in apoptosis can be alleviated, resulting in the selective killing of malignant cells. Here, we outline the progress of three small-molecule Bcl-2 antagonists that have advanced into clinical evaluation.
RESUMEN
Apoptosis is essential for normal development and maintenance of homeostasis, and disruption of apoptotic pathways is associated with multiple disease states, including cancer. Although initially identified as central regulators of apoptosis at the level of mitochondria, an important role for BCL-2 proteins at the endoplasmic reticulum is now well established. Signaling pathways emanating from the endoplasmic reticulum (ER) are involved in apoptosis initiated by stimuli as diverse as ER stress, oncogene expression, death receptor (DR) ligation and oxidative stress, and the BCL-2 family is almost invariably implicated in the regulation of these pathways. This also includes Ca(2+)-mediated cross talk between ER and mitochondria during apoptosis, which contributes to the mitochondrial dynamics that support the core mitochondrial apoptosis pathway. In addition to the regulation of apoptosis, BCL-2 proteins at the ER also regulate autophagy, a survival pathway that limits metabolic stress, genomic instability and tumorigenesis. In cases where apoptosis is inhibited, however, prolonged autophagy can lead to cell death. This review provides an overview of ER-associated apoptotic and autophagic signaling pathways, with particular emphasis on the BCL-2 family proteins.
Asunto(s)
Apoptosis , Autofagia , Retículo Endoplásmico/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Humanos , Estrés OxidativoRESUMEN
Chronic lymphocytic leukemia (CLL) is a B-cell lymphoid neoplasm with deregulated apoptosis and overexpression of several antiapoptotic BCL-2 proteins. GX15-070/Obatoclax is a small-molecule BH3 mimetic compound that has shown activity against several hematologic malignancies and solid tumors. In the present work, we report that GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells, followed by the activation of the mitochondrial apoptotic pathway. CLL cells showed lower sensitivity to GX15-070 than primary mantle cell lymphoma (MCL) ones, in correlation with higher levels of phosphorylated BCL-2 at serine 70 residue (pBCL-2(Ser70)) in CLL cells. Decrease in BCL-2 phosphorylation by extracellular signal-regulated kinase (ERK)1/2 inhibition increased CLL sensitivity to GX15-070, while blocking BCL-2 dephosphorylation using a PP2A antagonist reduced the activity of this BH3 mimetic. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of phosphorylated BCL-2, the degree of interaction between GX15-070 and bortezomib was regulated by basal pBCL-2(Ser70) levels. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácidos Borónicos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazinas/farmacología , Pirroles/farmacología , Apoptosis , Bortezomib , Sinergismo Farmacológico , Humanos , Indoles , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Mitocondrias/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction.
Asunto(s)
Proteínas Portadoras/metabolismo , Grupo Citocromo c/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina Endopeptidasas/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Muerte Celular , Citosol/metabolismo , Citotoxicidad Inmunológica , Granzimas , Humanos , Membranas Intracelulares/metabolismo , Células Jurkat/virología , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.
Asunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas/metabolismo , Receptor fas/metabolismo , Actomiosina/metabolismo , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , División Celular , Membrana Celular/metabolismo , Membrana Celular/patología , Humanos , Proteínas de la Membrana/genética , Mitocondrias/fisiología , Proteínas/genéticaRESUMEN
E1A and c-myc are oncogenes that can deregulate the cell cycle and promote transformation under conditions where normal cell-cycle checkpoints are inactivated. In situations where cell-cycle checkpoints are intact, the E1A and c-Myc proteins potently induce apoptosis, a property that is believed to be the end result of a cellular response to uncontrolled growth-promoting signals. p53 is a key regulator of E1A and c-myc-induced apoptosis and, together with the oncoproteins, may transcriptionally activate numerous genes whose products influence, or are themselves, members of the core apoptotic machinery. The upstream signaling events and the ultimate apoptotic pathways activated by E1A and c-Myc are discussed in this review.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Apoptosis/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Humanos , Transducción de Señal , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
We have cloned a 35-kDa protein from a mouse cDNA library with a 25% overall amino acid identity to yTom40 and 27% identity to nTom40. This homolog toTom40 was named MOM35. It contains two possible start codons 36 amino acids apart from each other. Both the long and the short version of MOM35 can be imported in vitro into mouse mitochondria. The identified protein is imported into the outer mitochondrial membrane and comprises a trypsin-resistance pattern similar to that of nTom40. Tom40 of N. crassa, S. cerevisiae, and the protein identified herein contains a highly conserved region with possible physiological importance. Subsequent investigation has revealed that this region interacts specifically in vitro with preproteins proposed to be imported by a Tom40-dependent pathway.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Biblioteca de Genes , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales , Datos de Secuencia Molecular , Neurospora crassa/genética , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tripsina/metabolismoRESUMEN
In the absence of an apoptotic signal, BAX adopts a conformation that constrains the protein from integrating into mitochondrial membranes. Here, we show that caspases, including caspase-8, can initiate BAX insertion into mitochondria in vivo and in vitro. The cleavage product of caspase-8, tBID, induced insertion of BAX into mitochondria in vivo, and reconstitution in vitro showed that tBID, either directly or indirectly, relieved inhibition of the BAX transmembrane signal-anchor by the NH2-terminal domain, resulting in integration of BAX into mitochondrial membrane. In contrast to these findings, however, Bid-null mouse embryo fibroblasts supported Bax insertion into mitochondria in response to death signaling by either TNFalpha or E1A, despite the fact that cytochrome c release from the organelle was inhibited. We conclude, therefore, that a parallel Bid-independent pathway exists in these cells for mitochondrial insertion of Bax and that, in the absence of Bid, cytochrome c release can be uncoupled from Bax membrane insertion.
Asunto(s)
Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/genética , Células Cultivadas , Grupo Citocromo c/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales , Fibroblastos/fisiología , Humanos , Ratones , Microscopía Confocal , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Ratas , Transducción de Señal/fisiología , Proteína X Asociada a bcl-2 , Receptor fas/metabolismoRESUMEN
Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent beta-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.
Asunto(s)
Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias Cardíacas/metabolismo , Porinas/metabolismo , Receptores de Superficie Celular , Animales , Transporte Biológico , Humanos , Membranas Intracelulares/metabolismo , Mitocondrias Cardíacas/ultraestructura , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Ratas , Canales Aniónicos Dependientes del VoltajeRESUMEN
Expression of the 243-residue form of the adenovirus E1A protein in the absence of other viral proteins triggers apoptosis by a pathway that requires p53. This pathway includes processing and activation of initiator procaspase-8, redistribution of cytochrome c, and activation of procaspase-3. Bcl-2 functions at or upstream of procaspase-8 processing to inhibit all of these events and prevent cell death. This contrasts with the anti-apoptotic influence of Bcl-2 family proteins in the cell death pathway induced by Fas ligand or tumor necrosis factor (TNF), in which Bcl-2 typically acts downstream of Fas/TNFR1-mediated activation of caspase-8. Moreover, E1A induces procaspase-8 processing and cell death in cells deleted of FADD, an adaptor protein critical for Fas/TNFR1 activation of caspase-8. The results indicate that E1A is capable of activating caspase-8 by a Bcl-2-inhibitable pathway that does not involve autocrine stimulation of FADD-dependent death receptor pathways.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Arabidopsis , Caspasas/metabolismo , Ácido Graso Desaturasas/metabolismo , Proteínas de Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Caspasa 8 , Caspasa 9 , Catálisis , Células Cultivadas , Grupo Citocromo c/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Mitocondrias/enzimología , Mitocondrias/metabolismoRESUMEN
Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.
Asunto(s)
Caspasas/biosíntesis , Grupo Citocromo c/metabolismo , Proteínas de la Membrana , Fotoquimioterapia , Proteínas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasa 6 , Caspasa 7 , Caspasa 8 , Caspasa 9 , Activación Enzimática , Precursores Enzimáticos/biosíntesis , Células HeLa , Humanos , Oligopéptidos/farmacologíaRESUMEN
The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Citosol/metabolismo , Humanos , Células KB , Masculino , Ratones , Datos de Secuencia Molecular , Miocardio/citología , Miocardio/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína X Asociada a bcl-2RESUMEN
Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.
Asunto(s)
Proteínas E4 de Adenovirus/fisiología , Adenovirus Humanos/fisiología , Apoptosis , Proteína p53 Supresora de Tumor/metabolismo , Proteínas E4 de Adenovirus/genética , Adenovirus Humanos/genética , Animales , Línea Celular , Línea Celular Transformada , Cricetinae , Expresión Génica , Células HeLa , Humanos , Ratones , Mutagénesis , Sistemas de Lectura Abierta , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Bap31 is a polytopic integral membrane protein of the endoplasmic reticulum and forms a complex with Bcl-2/Bcl-XL and procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D., Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol. 139, 327-338). In co-transfected human cells, procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule in Caenorhabditis elegans that binds to and activates the C. elegans procaspase, proCed-3. Here, we show that the predicted death effector homology domain within the cytosolic region of Bap31 interacts with Ced-4 and contributes to recruitment of procaspase-8. Bcl-XL, which binds directly but weakly to the polytopic transmembrane region of Bap31, indirectly and cooperatively associates with the Bap31 cytosolic domain, dependent on the presence of procaspase-8 and Ced-4. Ced-4Deltac does not interact with Bcl-XL but rather displaces it from Bap31, suggesting that an endogenous Ced-4-like adaptor is a normal constituent of the Bap31 complex and is required for stable association of Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a core death regulatory machinery.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/metabolismo , Caspasas , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas del Helminto/metabolismo , Proteínas de la Membrana , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Caspasa 8 , Caspasa 9 , Línea Celular , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteína bcl-XRESUMEN
In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207-6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J. G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529-535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-XL. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.
Asunto(s)
Proteínas E4 de Adenovirus/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis , Caspasas , Proteína p53 Supresora de Tumor/fisiología , Proteínas E4 de Adenovirus/genética , Animales , Apoptosis/efectos de los fármacos , Células CHO , Caspasa 3 , Línea Celular , Membrana Celular/química , Membrana Celular/ultraestructura , Núcleo Celular/ultraestructura , Cricetinae , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Doxiciclina/farmacología , Expresión Génica , Genes Reporteros , Genes bcl-2 , Membranas Intracelulares/fisiología , Potenciales de la Membrana , Ratones , Mitocondrias/fisiología , Fosfatidilserinas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
This study was undertaken to identify novel mitochondrial membrane proteins that are potential targets for phosphorylation. Mitochondrial membranes were incubated in the presence of [gamma-32P]ATP and the Triton X-114 extractable protein was subjected to ion-exchange and polyacrylamide gel chromatography to purify a major phosphorylated protein of approximately 17000 Da. The determined peptide sequence of the purified phosphoprotein corresponded to a segment of cytochrome c oxidase subunit IV, an inner membrane protein of 17160 Da. The identity of the phosphoprotein was confirmed by two-dimensional electrophoresis and Western blotting. The results identify mitochondrial cytochrome c oxidase subunit IV as a protein which is phosphorylated by an endogenous kinase.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias Cardíacas/enzimología , Mitocondrias Hepáticas/enzimología , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía por Intercambio Iónico , Perros , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/aislamiento & purificación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Membranas Intracelulares/enzimología , Microsomas/enzimología , Datos de Secuencia Molecular , Páncreas/enzimología , Radioisótopos de Fósforo/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de la Membrana , Microsomas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Caspasa 1 , Clonación Molecular , Cricetinae , Cisteína Endopeptidasas/aislamiento & purificación , Precursores Enzimáticos/aislamiento & purificación , Glutatión Transferasa , Humanos , Células KB , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase and conditions established to measure specific interactions of the receptor component with precursor proteins in vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Precursores de Proteínas/metabolismo , Receptores de Superficie Celular , Adenosina Trifosfato/metabolismo , Anticuerpos/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Detergentes/farmacología , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Etilmaleimida/metabolismo , Humanos , Técnicas In Vitro , Canales Iónicos , Proteínas de la Membrana/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales , Modelos Moleculares , Mutación , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Cloruro de Sodio/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Proteína Desacopladora 1RESUMEN
Tom70p is targeted and inserted into the mitochondrial outer membrane in the Nin-Ccyto orientation, via an NH2-terminal signal anchor sequence. The signal anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10) followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal domain with a matrix-targeting signal caused the signal anchor to adopt the reverse orientation in the outer membrane (Ncyto-Cin) or, if presented to mitoplasts, to arrest protein translocation at the inner membrane without insertion. Physically separating the transmembrane segment from the matrix-targeting signal by moving it downstream within the protein resulted in a failure to arrest in either membrane, and consequently the protein was imported to the matrix. However, if the mean hydrophobicity of the Tom70p transmembrane segment was increased in these constructs, the protein inserted into the inner membrane with an Nin-Cout orientation. Therefore we have determined conditions that allow the Tom70p transmembrane domain to insert in either membrane, pass through both membranes, or arrest without insertion in the inner membrane. These results identify the mean hydrophobicity of potential transmembrane domains within bitopic proteins as an important determinant for insertion into the mitochondrial inner membrane.