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1.
Nucleic Acids Res ; 50(14): 8207-8225, 2022 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-35848924

RESUMEN

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.


Asunto(s)
Melanoma , Metástasis de la Neoplasia , Proteína Disulfuro Isomerasas , Proteínas de Unión al ARN , Línea Celular Tumoral , Retículo Endoplásmico , Humanos , Melanoma/genética , Melanoma/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Metástasis de la Neoplasia/genética , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
2.
RNA ; 27(2): 190-201, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33172965

RESUMEN

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms. We find that CIRBP transcript levels correlate with breast cancer subtype and are an indicator of luminal A/B prognosis. Accordingly, overexpression of CIRBP in nontumoral MCF-10A cells promotes cell growth and clonogenicity, while depletion of CIRBP from luminal A MCF-7 cells has opposite effects. We use RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to identify a set of 204 high confident CIRBP targets in MCF-7 cells. About 10% of these showed complementary changes after CIRBP manipulation in MCF-10A and MCF-7 cells, and were highly interconnected with known breast cancer genes. To test the potential of CIRBP-mediated regulation of these targets in breast cancer development, we focused on Cystatin C (CST3), one of the most highly interconnected genes, encoding a protein that displays tumor suppressive capacities. CST3 depletion restored the effects of CIRBP depletion in MCF-7 cells, indicating that CIRBP functions, at least in part, by down-regulating CST3 levels. Our data provide a resource of CIRBP targets in breast cancer, and identify CST3 as a novel downstream mediator of CIRBP function.


Asunto(s)
Neoplasias de la Mama/genética , Cistatina C/genética , Regulación Neoplásica de la Expresión Génica , Glándulas Mamarias Humanas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Cistatina C/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Redes Reguladoras de Genes , Humanos , Glándulas Mamarias Humanas/patología , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Análisis de Supervivencia
3.
RNA ; 24(4): 529-539, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29317541

RESUMEN

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.


Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Poliadenilación/fisiología , Polinucleotido Adenililtransferasa/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/química , Ribonucleasa III/metabolismo , Receptores Toll-Like/química , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Polinucleotido Adenililtransferasa/genética , Biosíntesis de Proteínas/genética , Señales de Poliadenilación de ARN 3'/genética , ARN Helicasas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Factores de Escisión y Poliadenilación de ARNm/genética
4.
Nucleic Acids Res ; 46(8): 4099-4113, 2018 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-29635389

RESUMEN

Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Regulación de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Factores de Transcripción/genética , Regiones no Traducidas 5' , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factor 3 de Iniciación Eucariótica/antagonistas & inhibidores , Femenino , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Transcripción/metabolismo
5.
Genes Dev ; 24(2): 129-34, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20080951

RESUMEN

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation that requires two sequences in the 3' untranslated region (UTR) of vertebrate substrates: the polyadenylation hexanucleotide, and the cytoplasmic polyadenylation element (CPE). Using a cell-free Drosophila system, we show that these signals are not relevant for Toll polyadenylation but, instead, a "polyadenylation region" (PR) is necessary. Competition experiments indicate that PR-mediated polyadenylation is required for viability and is mechanistically distinct from the CPE/hexanucleotide-mediated process. These data indicate that Toll mRNA is polyadenylated by a noncanonical mechanism, and suggest that a novel machinery functions for cytoplasmic polyadenylation during Drosophila embryogenesis.


Asunto(s)
Citoplasma/metabolismo , Drosophila melanogaster/embriología , Poliadenilación/fisiología , Regiones no Traducidas 3' , Animales , Proteínas de Drosophila/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo
6.
Life Sci Alliance ; 5(12)2022 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-36114004

RESUMEN

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative noncanonical, CPEB-independent complex in <i>Drosophila</i>, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein cofactors in cytoplasmic polyadenylation. Using RIP-Seq analysis, we identify hundreds of potential Dicer-2 target transcripts, ∼60% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA-independent complex with Dicer-2 and mediate interactions of Dicer-2 with Wispy. Functional poly(A)-test analyses showed that Twenty-four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer-2 targets. Our results reveal components of a novel cytoplasmic polyadenylation complex that operates during <i>Drosophila</i> early embryogenesis.


Asunto(s)
Ataxina-2 , Poliadenilación , Animales , Ataxina-2/genética , Ataxina-2/metabolismo , Drosophila/genética , Drosophila/metabolismo , Poliadenilación/genética , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
7.
Cell Rep ; 38(2): 110211, 2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-35021076

RESUMEN

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.


Asunto(s)
Senescencia Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/fisiología , Línea Celular , Proliferación Celular/fisiología , Senescencia Celular/genética , Proteínas de Unión al ADN/fisiología , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Humanos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Oncogenes/genética , Cultivo Primario de Células , Procesamiento Postranscripcional del ARN/fisiología , Proteínas de Unión al ARN/fisiología , Fenotipo Secretor Asociado a la Senescencia/genética , Fenotipo Secretor Asociado a la Senescencia/fisiología , Transducción de Señal/fisiología , Proteína 1 de Unión a la Caja Y/metabolismo
8.
FEBS Lett ; 526(1-3): 15-20, 2002 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-12208496

RESUMEN

This study examined the role of ceramide generated by exogenous sphingomyelinases (SMases) on transcription nuclear factor-kappa B (NF-kappa B) activation and apoptosis in human colon epithelial HT-29 cells. Exogenous neutral (N) and acidic (A) SMase activated NF-kappa B with different kinetics, accounting for the diverse pattern of DNA binding of NF-kappa B complexes activated by tumor necrosis factor-alpha (TNF). NSMase activated predominantly RelA/p52 and RelA/p50 dimers within 30 min, while ASMase activated the p50/p50 homodimer by 20 h. The predominant activation of RelA-containing kappa B complexes by TNF or NSMase paralleled the induction of interleukin-8. HT-29 cells were sensitive to ASMase and TNF but resistant to NSMase. However, the apoptotic potential of NSMase was masked by NF-kappa B, as its prior inactivation sensitized HT-29 cells to NSMase. Thus, the generation of ceramide by exogenous SMases participates differentially in inflammation and apoptosis.


Asunto(s)
Apoptosis/fisiología , Ceramidas/fisiología , Mucosa Intestinal/fisiología , FN-kappa B/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Neoplasias del Colon , Dimerización , Humanos , Cinética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología
9.
Methods Mol Biol ; 1125: 53-63, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24590779

RESUMEN

Basic research in Drosophila melanogaster has benefited from a plethora of powerful genetics tools. Detailed biochemical analysis, however, has often been difficult due to the lack of in vitro systems that faithfully recapitulate the observations made in vivo. In the field of posttranscriptional regulation, the recent establishment of robust in vitro systems from embryo and ovary material has fueled the mechanistic understanding of a variety of processes. Here we describe protocols to obtain and use extracts from Drosophila embryos that are competent for cytoplasmic polyadenylation and translation of exogenously added transcripts.


Asunto(s)
Bioensayo/métodos , Citoplasma/metabolismo , Poliadenilación/fisiología , Animales , Drosophila/metabolismo , Femenino , Masculino , Ovario/metabolismo
10.
Curr Opin Genet Dev ; 21(4): 452-7, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21536428

RESUMEN

Cytoplasmic polyadenylation is the process by which dormant, translationally inactive mRNAs become activated via the elongation of their poly(A) tails in the cytoplasm. This process is regulated by the conserved cytoplasmic polyadenylation element binding (CPEB) protein family. Recent studies have advanced our understanding of the molecular code that dictates the timing of CPEB-mediated poly(A) tail elongation and the extent of translational activation. In addition, evidence for CPEB-independent mechanisms has accumulated, and the breath of biological circumstances in which cytoplasmic polyadenylation plays a role has expanded. These observations underscore the versatility of CPEB as a translational regulator, and highlight the diversity of cytoplasmic polyadenylation mechanisms.


Asunto(s)
Citoplasma/metabolismo , Poliadenilación/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Activación Transcripcional
11.
J Lipid Res ; 48(9): 1924-35, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17556754

RESUMEN

Ceramide regulates many cellular processes, including cell growth, differentiation, and apoptosis. Although the effects of exogenous bacterial neutral sphingomyelinase (SMase) in Xenopus laevis oocytes have been investigated, its microinjection into oocytes has not been reported previously. Thus, we compared the incubation versus microinjection of the neutral Bacillus cereus sphingomyelinase (bSMase) to examine whether the topology of ceramide generation determines its effects on the fate of oocytes. In agreement with previous findings, incubation of mature stage VI oocytes with bSMase increased ceramide levels in oocyte extracts over time, causing the germinal vesicle breakdown indicative of maturation, without evidence of cytotoxicity. In contrast, bSMase microinjection, which increased ceramide levels in a time- and dose-dependent manner, resulted in oocyte apoptosis characterized by reactive oxygen species (ROS) generation, reduced glutathione (GSH) depletion in cytosol and mitochondria, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Microinjection of acidic SMase from human placenta recapitulated the apoptotic effects of bSMase microinjection. Preincubation of oocytes with GSH-ethyl ester before bSMase microinjection prevented ROS generation and mitochondrial downstream events, thus protecting oocytes from bSMase-induced death. These findings show a divergent action of bSMase-induced ceramide on oocyte maturation or apoptosis depending on the intracellular site where ceramide is generated.


Asunto(s)
Apoptosis/efectos de los fármacos , Ceramidas/fisiología , Oocitos/efectos de los fármacos , Esfingomielina Fosfodiesterasa/fisiología , Animales , Bacillus cereus/enzimología , Ceramidas/farmacología , Femenino , Xenopus laevis
12.
Genes Dev ; 20(3): 380-9, 2006 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-16452509

RESUMEN

The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (SXL) is an essential regulatory step for X-chromosome dosage compensation in Drosophila melanogaster. The mammalian upstream of N-ras (UNR) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. Here we have identified the Drosophila homolog of mammalian UNR as a cofactor required for SXL-mediated repression of msl-2 translation. UNR interacts with SXL, a female-specific protein. Although UNR is present in both male and female flies, binding of SXL to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits UNR to adjacent regulatory sequences, thereby conferring a sex-specific function to UNR. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with SXL in translational control.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Compensación de Dosificación (Genética) , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila/genética , Proteínas Nucleares/fisiología , Biosíntesis de Proteínas/fisiología , Factores de Transcripción/fisiología , Cromosoma X/metabolismo , Regiones no Traducidas 3'/metabolismo , Animales , Proteínas de Unión al ADN/genética , Compensación de Dosificación (Genética)/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Masculino , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo
13.
Hepatology ; 38(3): 692-702, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12939596

RESUMEN

The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 +/- 0.3 mmol/L and maximum velocity of 1.9 +/- 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A(2)C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A(2)C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion.


Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Proteínas Portadoras/metabolismo , Glutatión/deficiencia , Proteínas de Transporte de Membrana , Mitocondrias Hepáticas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Colesterol/farmacología , Glutatión/metabolismo , Humanos , Masculino , Fluidez de la Membrana , Oocitos , Ratas , Ratas Sprague-Dawley , Xenopus laevis
14.
J Biol Chem ; 277(51): 49870-6, 2002 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-12351644

RESUMEN

Ganglioside GD3 (GD3) has emerged as a modulator of cell death pathways due to its ability to interact with mitochondria and disable survival pathways. Because NF-kappaB activation contributes to cancer therapy resistance, this study was undertaken to test whether GD3 modulates the response of human hepatoblastoma HepG2 cells to radio- and chemotherapy. NF-kappaB was activated in HepG2 cells shortly after therapeutic doses of ionizing radiation or daunorubicin treatment that translated into up-regulation of kappaB-dependent genes. These effects were accompanied by minimal killing of HepG2 cells by either ionizing radiation or daunorubicin. However, GD3 pretreatment blocked the nuclear translocation of active kappaB members, without effect on Akt phosphorylation, induced by either treatment. The suppression of kappaB-dependent gene induction by GD3 was accompanied by enhanced apoptotic cell death caused by these therapies. Furthermore, the combination of GD3 plus ionizing radiation stimulated the formation of reactive species followed by the mitochondrial release of cytochrome c and Smac/Diablo and caspase 3 activation. Pretreatment with cyclosporin A before radiotherapy protected HepG2 cells from the therapeutic combination of GD3 plus ionizing radiation. These findings underscore a key role of mitochondria in the response of tumor cells to cancer therapy and highlight the potential relevance of GD3 to overcome resistance to cancer therapy by combining its dual action as a mitochondria-interacting and NF-kappaB-inactivating agent.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/radioterapia , Gangliósidos/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/radioterapia , Proteínas Serina-Treonina Quinasas , Apoptosis , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Confocal , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Genéticos , FN-kappa B/metabolismo , Fosforilación , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Fármacos Sensibilizantes a Radiaciones/farmacología , Especies Reactivas de Oxígeno , Transducción de Señal , Factores de Tiempo , Factor de Transcripción ReIA , Activación Transcripcional , Regulación hacia Arriba
15.
J Biol Chem ; 278(36): 33928-35, 2003 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-12821666

RESUMEN

Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.


Asunto(s)
Translocador 1 del Nucleótido Adenina/metabolismo , Colesterol/fisiología , Adenina/química , Translocador 1 del Nucleótido Adenina/química , Animales , Apoptosis , Atractilósido/química , Atractilósido/farmacología , Transporte Biológico , Western Blotting , Muerte Celular , Colesterol/metabolismo , Grupo Citocromo c/metabolismo , Inhibidores Enzimáticos/farmacología , Microscopía Electrónica , Mitocondrias/metabolismo , Mitocondrias Hepáticas/metabolismo , Permeabilidad , Fosfolípidos/metabolismo , Conformación Proteica , Ratas , Factores de Tiempo
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