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1.
Proc Natl Acad Sci U S A ; 108(41): 17213-8, 2011 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-21969592

RESUMEN

Stress has been identified as a major causal factor for many mental disorders. However, our knowledge about the chain of molecular and cellular events translating stress experience into altered behavior is still rather scant. Here, we have characterized a murine ortholog of the putative tumor suppressor gene DRR1 as a unique stress-induced protein in brain. It binds to actin, promotes bundling and stabilization of actin filaments, and impacts on actin-dependent neurite outgrowth. Endogenous DRR1 localizes to some, but not all, synapses, with preference for the presynaptic region. Hippocampal virus-mediated enhancement of DRR1 expression reduced spine density, diminished the probability of synaptic glutamate release, and altered cognitive performance. DRR1 emerges as a protein to link stress with actin dynamics, which in addition is able to act on synaptic function and cognition.


Asunto(s)
Cognición/fisiología , Sinapsis/fisiología , Proteínas Supresoras de Tumor/fisiología , Actinas/metabolismo , Animales , Conducta Animal/fisiología , Encéfalo/citología , Encéfalo/fisiología , Genes Supresores de Tumor , Células HEK293 , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuritas/metabolismo , Neuritas/ultraestructura , Unión Proteica , Estrés Fisiológico , Proteínas Supresoras de Tumor/genética
2.
Mol Endocrinol ; 16(6): 1352-66, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12040020

RESUMEN

To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.


Asunto(s)
Fase G2 , Mitosis , Receptores de Glucocorticoides/metabolismo , Animales , Bencimidazoles/farmacología , Células CHO , Línea Celular , Cricetinae , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Citometría de Flujo , Virus del Tumor Mamario del Ratón/genética , Ratones , Regiones Promotoras Genéticas/genética , Moldes Genéticos , Activación Transcripcional/efectos de los fármacos , Transfección , Células Tumorales Cultivadas , Tirosina Transaminasa/genética , Tirosina Transaminasa/metabolismo
3.
Mol Endocrinol ; 17(10): 1991-2001, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12869591

RESUMEN

Cisplatin is an antineoplastic drug that binds to DNA, thereby inhibiting cell division and tumor growth. Cisplatin may also disrupt the function of some proteins, including heat shock protein 90 (Hsp90). We report that cisplatin dose-dependently inhibited transcriptional activity of the androgen receptor and the glucocorticoid receptor (GR) in transient reporter assays. A truncated, hormone-independent GR was only partially inhibited at significantly higher doses of cisplatin. Cisplatin treatment of neuroblastoma cells led to an immediate inhibition of hormone binding by GR, followed by proteasome-dependent degradation of the receptor. Other Hsp90-regulated proteins, i.e. the phosphokinases raf-1, lck, and c-src, were not affected, indicating a specific functional interference of cisplatin with the steroid receptors GR and androgen receptor. Cisplatin did not elicit a stress response, in contrast to geldanamycin. Immunoprecipitation revealed that cisplatin disrupts binding of GR to Hsp90. Moreover, cisplatin-treated Hsp90 was unable to associate with untreated ligand binding domain of GR. Reticulocyte lysate was able to restore hormone binding of GR in vitro, but not when the lysate was pretreated with geldanamycin. Our data reveal that cisplatin influences steroid receptors also independently of its DNA-mediated effects and, thus, suggest a novel modes of action for this cytostatic drug.


Asunto(s)
Antagonistas de Receptores Androgénicos , Cisplatino/farmacología , ADN/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Antineoplásicos/farmacología , Benzoquinonas , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Lactamas Macrocíclicas , Luciferasas/metabolismo , Quinonas/farmacología , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Reticulocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección
4.
FEBS Lett ; 560(1-3): 35-8, 2004 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-14987994

RESUMEN

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Sustitución de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Clonales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Luciferasas/metabolismo , Proteínas Luminiscentes , Neuroblastoma , Mutación Puntual , Pruebas de Precipitina , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Transfección
5.
J Biol Chem ; 280(6): 4609-16, 2005 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-15591061

RESUMEN

We used a cellular system to elucidate the molecular determinants of the large immunophilin FK506-binding proteins (FKBP)51 and -52 for their action on the glucocorticoid receptor in mammalian cells. Increasing the levels of FKBP51 reduced the transcriptional activity of the receptor, as reported. Elevated levels of FKBP52 per se showed no effect but mitigated the inhibition of the receptor induced by FKBP51. We discovered that nuclear translocation of the glucocorticoid receptor was delayed by FKBP51. This correlates with the reduced interaction of FKBP51 with the motor protein dynein compared with FKBP52. From mutational analyses, we concluded that three features of the immunophilins are required for efficient receptor signaling in mammalian cells: hsp90 interaction, dynein association, and peptidylprolyl isomerase (PPIase) enzyme activity. The relevance of dynein for receptor function was substantiated by several experiments: 1) coexpression of dynamitin, which disrupts the transport complex and reduces receptor activity; 2) coexpression of the PPIase domain fragment of FKBP52, which is known to disrupt interaction of the receptor to dynein and reduce glucocorticoid receptor function, in contrast to the corresponding fragment of FKBP51; and 3) swapping of the PPIase domains FKBP51 and FKBP52, which reverses the respective activity. We concluded from our results that the mechanisms of the regulatory system FKBP51/FKBP52 discovered in yeast also operate in mammals to modulate hormone binding of the receptor. In addition, differential regulation of dynein association and nuclear translocation contributes to the effects of the two immunophilins on the glucocorticoid receptor in mammals.


Asunto(s)
Receptores de Glucocorticoides/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/fisiología , Tacrolimus/farmacología , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Análisis Mutacional de ADN , Dineínas/química , Glucocorticoides/química , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Inmunofilinas/química , Inmunoprecipitación , Luciferasas/metabolismo , Isomerasa de Peptidilprolil/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión a Tacrolimus/metabolismo , Factores de Tiempo , Transfección , Tirosina/química , beta-Galactosidasa/metabolismo
6.
J Biol Chem ; 278(7): 4926-31, 2003 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-12482863

RESUMEN

The co-chaperone BAG-1 is involved in the regulation of steroid hormone receptors, including the glucocorticoid receptor (GR). More recently, BAG-1 was found in the nucleus where it decreases GR transactivation. Moreover, nonspecific DNA binding of BAG-1 has been reported. We discovered that of the N-terminal part of BAG-1M, the first 8 amino acids are sufficient for DNA binding, containing a stretch of three lysines and a stretch of three arginines. Changing the spacing between these stretches had no effect on DNA binding. Surprisingly, this small, nonsequence-specific DNA binding domain was nonetheless necessary for the inhibitory function of BAG-1 for GR-dependent transcription, whereas the following serine- and threonine-rich E(2)X(4) repeat domain was not. Mutational analysis of these two domains revealed that only mutants retaining DNA binding capability were able to down-regulate GR-mediated transactivation. Intriguingly, lack of DNA binding could not be functionally rescued by BAG-1M harboring a point mutation abolishing interaction with hsp70. Thus, DNA binding and hsp70 interaction are required in cis. We propose that the nonsequence-specific DNA-binding protein BAG-1 acts at specific chromosomal loci by interacting with other proteins.


Asunto(s)
Proteínas Portadoras/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Sitios de Unión/genética , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Mutación , Unión Proteica , Estructura Terciaria de Proteína/genética , Receptores de Glucocorticoides/química , Transducción de Señal , Factores de Transcripción
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