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1.
Cell Death Differ ; 15(5): 859-66, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18239673

RESUMEN

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.


Asunto(s)
Diferenciación Celular/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/fisiología , Proteínas HSP90 de Choque Térmico/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Macrófagos/citología , Macrófagos/fisiología , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
2.
FEMS Microbiol Rev ; 23(5): 551-61, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10525166

RESUMEN

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.


Asunto(s)
Transducción de Señal , Toxoplasma/patogenicidad , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Movimiento Celular , Proteínas de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Toxoplasma/fisiología
3.
Eur J Cell Biol ; 74(1): 92-101, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9309395

RESUMEN

The tachyzoite of Toxoplasma gondii must successfully invade a host cell before it can replicate. Depletion of the Ca2+ in the external medium (EGTA) reduced tachyzoite invasion, suggesting that the initial tachyzoite-host cell interaction is Ca2+ dependent. The interaction of tachyzoites with host cells was also inhibited by Ca2+ channel blockers (verapamil) and calmodulin antagonists (trifluoperazine, calmidazolium). The calmodulin concentrated at the apical end of the tachyzoite could be involved in cytoskeleton movement and conoid extrusion. Invasion also depends on changes in tachyzoite cytosolic calcium. Depletion of Ca2+ with A23187+EGTA and release of Ca2+ from intratachyzoite pools (nuclear and perinuclear areas) inhibited invasion. In contrast, Ca-ionophore and thapsigargin which increase host cell cytosolic Ca2+, significantly decreased tachyzoite invasion. We therefore suggest that the effect of the drug is significantly different from the localized Ca2+ signal that is produced after parasite attachment to its host cell receptors and leads to its internalization into the host cell.


Asunto(s)
Calcio/fisiología , Calmodulina/fisiología , Toxoplasma/patogenicidad , Animales , Calcimicina/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Calmodulina/análisis , Calmodulina/antagonistas & inhibidores , Carcinoma de Células Escamosas , Citosol/metabolismo , Células Epiteliales/parasitología , Humanos , Imidazoles/farmacología , Ionóforos/farmacología , Trifluoperazina/farmacología , Células Tumorales Cultivadas , Verapamilo/farmacología
4.
Int J Parasitol ; 31(10): 1114-20, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11429176

RESUMEN

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.


Asunto(s)
Monocitos/fisiología , Monocitos/parasitología , Toxoplasma/fisiología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/fisiología , Animales , Carbocianinas/farmacología , Membrana Celular/fisiología , Diciclohexilcarbodiimida/farmacología , Inhibidores Enzimáticos/farmacología , Gramicidina/farmacología , Humanos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Microscopía Fluorescente , Monocitos/inmunología , Omeprazol/farmacología , Ouabaína/farmacología , Vanadatos/farmacología
5.
Oncogene ; 28(37): 3332-44, 2009 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-19597476

RESUMEN

Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinas/metabolismo , Animales , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP27/química , Células HeLa , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Especificidad por Sustrato , Activación Transcripcional
6.
Cell Death Differ ; 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25361076

RESUMEN

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90ß. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90ß isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its ß isoform as specific depletion of HSP90α does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90ß both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90ß prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.Cell Death and Differentiation advance online publication, 1 February 2008; doi:10.1038/sj.cdd.4402320.

7.
J Biomed Mater Res ; 58(3): 238-46, 2001 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-11319736

RESUMEN

Hydroxyapatite used as bone replacement can lead to particle release in the implantation site. These particles interact with monocytes, which are the first immune cells to colonize the implant and an inflammatory site. Thanks to cryo-X-ray microanalysis, we can observe cells in a state close to the physiological one and we have access to diffusible ions. We paid particular attention to the potassium-to-sodium ratio, which is one of the best viability criteria. We used this method to study the interaction between three hydroxyapatite particles treated at three different temperatures (not treated, treated at 600 degrees C and 1180 degrees C), and monocytes. In the culture condition, the hydroxyapatite treated at 1180 degrees C underwent the least dissolution. We demonstrate that monocytes were altered by the three hydroxyapatite particles. The hydroxyapatite particules treated at 600 degrees C were found to be more toxic.


Asunto(s)
Sustitutos de Huesos/toxicidad , Durapatita/toxicidad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Sustitutos de Huesos/aislamiento & purificación , Calcio/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoplasma/metabolismo , Durapatita/aislamiento & purificación , Microanálisis por Sonda Electrónica , Calor , Humanos , Iones , Ensayo de Materiales , Microscopía Electrónica , Monocitos/ultraestructura , Tamaño de la Partícula , Fósforo/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Células U937
8.
Parasitol Res ; 85(10): 809-18, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10494806

RESUMEN

The invasion of host cells by the obligate intracellular protozoan parasite Toxoplasma gondii is calcium dependent. We have identified two calcium storage areas in tachyzoites, the endoplasmic reticulum and vesicles that contain high concentrations of calcium as amorphous calcium phosphate precipitates. Our data indicate that these vesicles slowly lose their calcium during the intracellular development of the tachyzoite as their nucleus phosphorus content increases. We found fluctuations in the sulfur content of the tachyzoite during invasion following the exocytosis of protein from the secretory organelles, with a loss of sodium and chlorine, and the uptake of potassium from the host cell cytoplasm. We demonstrated that penetration of the tachyzoite into the host cell was accompanied by increases in the concentrations of phosphorus and sulfur in the host cell nucleus, probably due to increased transcription. The cytosol sodium concentrations decreased, while the potassium content increased. Thus, the subcellular element distribution of tachyzoites and host cells changes during invasion and intracellular growth of the parasites. In addition, our results indicate that tachyzoite calcium might be involved in the egress of the parasite from the host cell.


Asunto(s)
Calcio/metabolismo , Monocitos/parasitología , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Animales , Crioultramicrotomía , Citosol/metabolismo , Microanálisis por Sonda Electrónica , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Ratones , Microscopía Electrónica de Transmisión de Rastreo , Monocitos/metabolismo , Monocitos/ultraestructura , Orgánulos/ultraestructura , Fósforo/metabolismo , Transducción de Señal , Azufre/metabolismo , Toxoplasma/ultraestructura , Células Tumorales Cultivadas , Vacuolas/ultraestructura
9.
Cell Struct Funct ; 26(1): 49-60, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11345503

RESUMEN

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.


Asunto(s)
ATPasas Transportadoras de Calcio/análisis , ATPasas Transportadoras de Calcio/metabolismo , Toxoplasma/enzimología , Adenosina Trifosfato/metabolismo , Animales , Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Microanálisis por Sonda Electrónica , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Magnesio/farmacología , Microscopía Electrónica , Microscopía Inmunoelectrónica , Potasio/farmacología , Tapsigargina/farmacología , Toxoplasma/ultraestructura , Vanadatos/farmacología
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