RESUMEN

The androgen receptor (AR) acts as an androgen-dependent transcription factor controlling the development of prostate tissue. Upon binding to androgen, AR undergoes a dynamic structural change leading to interaction between the NH(2)- and COOH-terminal regions of AR (N-C interaction). ARA24/Ran, which is a small GTPase, functions as an AR coactivator. Here, we report that ARA24/Ran enhances the N-C interaction of AR. The constitutively GTP- or GDP-bound form of ARA24/Ran repressed the AR N-C interaction. ARA24/Ran did not enhance the transcriptional activities of AR mutants that disrupt the N-C interaction. ARA24/Ran formed an endogenous protein complex with nuclear AR, but not cytoplasmic AR. Unlike SRC-1 with the positive activity for AR N-C interaction, ARA24/Ran did not enhance the transcriptional activity of the COOH-terminal domain-deleted AR mutant that is constitutively localized in the nucleus. These data demonstrate that ARA24/Ran increases AR transactivation by enhancing the AR N-C interaction in the nucleus.

Asunto(s)

Andrógenos/metabolismo , Receptores Androgénicos/metabolismo , Proteína de Unión al GTP ran/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Masculino , Mutación , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Transcripción Genética , Activación Transcripcional , Proteína de Unión al GTP ran/genética

RESUMEN

We evaluated the effectiveness of using Flinders Technology Associates (FTA) filter paper for the polymerase chain reaction (PCR) genotyping of transgenic mice. Tail prick blood sample dried on an FTA filter disc was processed for genomic PCR. It is easy and rapid to prepare DNA templates because the protocol is extraction-free and only requires minimal handling of wash briefly bloodstained FTA filter discs. Progeny of a transgene-positive founder mated with wild-type mice was screened for the presence of the transgene by the filter-based PCR using transgene-specific primers. The resulting amplicons with expected sizes of 3134 bp, 1152 bp, 877 bp and 688 bp were robust and reproducible, allowing a distinction between transgenic (n=44) and wild-type (n=47) mice showing no signal. The filter-based PCR screening took only half a day. The present study confirmed the validity and usefulness of the novel rapid extraction-free genotyping method.

Asunto(s)

Fenómenos Fisiológicos Sanguíneos , Genotipo , Filtros Microporos , Reacción en Cadena de la Polimerasa/métodos , Manejo de Especímenes/métodos , Transgenes , Animales , Cartilla de ADN , Técnicas Genéticas , Ratones , Ratones Transgénicos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo

RESUMEN

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).

Asunto(s)

Acetil-CoA Hidrolasa/genética , Acetil-CoA Hidrolasa/aislamiento & purificación , Citosol/enzimología , Expresión Génica , Acetil-CoA Hidrolasa/química , Acetil-CoA Hidrolasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Biblioteca de Genes , Humanos , Hígado/enzimología , Ratones , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Spodoptera

RESUMEN

Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn(2+)-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.

Asunto(s)

Extractos Celulares/farmacología , Receptores de Glucocorticoides/genética , Secuencias Reguladoras de Ácidos Nucleicos , Timosina/análogos & derivados , Transcripción Genética , Animales , Células COS , Proteína de Unión a CREB/metabolismo , Extractos Celulares/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteína p300 Asociada a E1A/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Histona Acetiltransferasas , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Coactivador 1 de Receptor Nuclear , Plásmidos , ARN Interferente Pequeño/farmacología , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Timosina/metabolismo , Factores de Transcripción/metabolismo , Tirosina Transaminasa/metabolismo , Proteína Fluorescente Roja

RESUMEN

In this report, we describe a new purification method for activated recombinant glucocorticoid receptor (GR) utilizing a cation-exchanger (Mono S) at pH8.4. This method is based upon a new finding that activated GR binds to both Mono Q and Mono S columns at the same pH. This method enables us to purify recombinant GR within 3 h. The purified GR represents more than 97% of the eluted proteins. Purified recombinant GR is able to bind specifically to a DNA fragment containing the glucocorticoid response element. Recombinant GR has no tag sequence that can be utilized for purification. Thus, this separation method is also applicable to purification of native GR.

Asunto(s)

Receptores de Glucocorticoides/aislamiento & purificación , Animales , Electroforesis en Gel de Poliacrilamida , Ratas , Proteínas Recombinantes/aislamiento & purificación , Spodoptera

RESUMEN

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (K(av)=0.47 on Superdex 200) and second of different size from that of the oligomeric form (K(av)=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism.

Asunto(s)

Acetil-CoA Hidrolasa/aislamiento & purificación , Cromatografía en Gel/métodos , Hígado/enzimología , Acetil-CoA Hidrolasa/química , Animales , Biopolímeros , Citosol/enzimología , Ratas

RESUMEN

We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.

Asunto(s)

Receptores de Glucocorticoides/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Certain amines known to be concentrated in lysosomes, termed "lysosomotropic amines," cause the formation of lysosomal vacuoles. A cell-free system was established to examine the effects of basic substances and acidic ionophores. In this system, the drugs not only increased the internal pH, but also caused a disruption of lysosomes. The osmotic swelling of lysosomes induced by protonated bases or cations for particular ionophores, which had accumulated within lysosomes driven by the proton pump, caused the osmotic lysis of lysosomes. The lysosomal disruption was inhibited upon the addition of the cytosol fraction. This phenomenon provides an in vitro system for studying the osmo-regulation and intercellular dynamics of the lysosomal system, including membrane fusion. The lysosomal stabilization factor was purified from the cytosol fraction and identified as ATP-stimulated glucocorticoid receptor translocation promoter (ASTP).

Asunto(s)

Adenosina Trifosfato/farmacología , Aminas/antagonistas & inhibidores , Aminas/farmacología , Citosol/química , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/farmacología , Lisosomas/efectos de los fármacos , Macrólidos , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Relación Dosis-Respuesta a Droga , Etilmaleimida/farmacología , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Hígado/química , Lisosomas/metabolismo , Masculino , Concentración Osmolar , Pruebas de Precipitina , Ratas , Ratas Wistar , Vacuolas

RESUMEN

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.

Asunto(s)

Acetil-CoA Hidrolasa/genética , Acetil-CoA Hidrolasa/metabolismo , Citosol/enzimología , Metabolismo de los Lípidos , Acetil-CoA Hidrolasa/química , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Hígado/enzimología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Spodoptera/citología