RESUMEN

BACKGROUND: Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). METHODS: Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. RESULTS: The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. CONCLUSIONS: Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.

Asunto(s)

Antineoplásicos/farmacología , Compuestos Azo/farmacología , Donantes de Óxido Nítrico/farmacología , Piperazinas/farmacología , Profármacos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Glutatión Transferasa/fisiología , Humanos , Inmunohistoquímica , Masculino , Óxido Nítrico/biosíntesis , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas

RESUMEN

Enhanced nuclear localization of nuclear factor κB (NF-κB) in prostate cancer (PCa) samples and constitutive NF-κB signaling in a class of PCa cell lines with low androgen receptor (AR) expression (PC3 and DU-145) imply an important role of the IκB kinase (IKK)/NF-κB system in PCa. However, most PCa and PCa cell lines depend on the activity of the AR, and the role of NF-κB in these AR-expressing PCa remains unclear. Here, we demonstrate that inhibition of NF-κB signaling by the IKK inhibitor BMS345541 reduced proliferation and increased apoptosis in AR-expressing PCa cell lines. Furthermore, AR activity and target gene expression were distinctively reduced, whereas AR protein levels remained unaltered on BMS345541 treatment. Similar effects were observed particularly after small interfering RNA (siRNA)-mediated knockdown of IKK1, but not by siRNA-mediated suppression of IKK2. Moreover, IKK1 overexpression augmented 5α-dihydrotestosterone-induced nuclear AR translocation, whereas nuclear AR was reduced by IKK1 knockdown or BMS345541. However, because IKK1 also enhances the activity of a chronically nuclear AR mutant, modulation of the subcellular distribution seems not to be the only mechanism by which IKK1 enhances AR activity. Finally, reduced in vivo AR phosphorylation after BMS345541 treatment and in vitro AR phosphorylation by IKK1 or IKK2 imply that AR constitutes a novel IKK target. Taken together, our data identify IKK1 as a potentially target structure for future therapeutic intervention in PCa.

Asunto(s)

Quinasa I-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Silenciador del Gen , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Imidazoles/farmacología , Masculino , Neoplasias de la Próstata/genética , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Quinoxalinas/farmacología , Transducción de Señal/efectos de los fármacos

RESUMEN

The molecular mechanisms leading to castration-resistant prostate cancer (CRPC) are poorly understood. Among several mechanisms leading to CRPC growth a dysregulation of androgen receptor (AR) co-regulators (i.e. up-regulation of co-activators or down-regulation of co-repressors) is discussed. There are numerous reports demonstrating an increased expression of co-activators during prostate cancer progression. On the contrary, the impact of co-repressors on tumor growth and development is less clear. In this study we compared the effects of two known co-repressors, NCoR and SMRT, on AR transcriptional activity in prostate cancer (PCa) cell lines and compared them to that in COS-1 cells. Interestingly, we found that NCoR/SMRT overexpression did not repress AR-dependent gene expression in the PCa cell lines, but rather activated it. This finding is probably due to an impaired AR-co-repressor interaction in the prostate cancer cell lines. In conclusion, we provide evidence that up-regulation of NCoR or SMRT may increase transcriptional activity of the AR in a cell type-specific context.

Asunto(s)

Co-Represor 2 de Receptor Nuclear/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Animales , Western Blotting , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Masculino , Co-Represor 2 de Receptor Nuclear/genética , Receptores Androgénicos/genética

RESUMEN

Nuclear localization of the ecdysteroid receptor (EcR) is increased in HeLa cells if exportin-1 (CRM1), a predominant carrier for export of proteins and RNA from the nucleus into the cytoplasm, is knocked down by siRNA against exportin. However, knockdown of the small G protein Ran, which is essential for nuclear transport, leads to an arrest of EcR in the cytoplasm, but does not prevent efficient nuclear import of the most important heterodimerization partner of EcR, ultraspiracle (Usp). Like in vertebrate cells, EcR is also distributed heterogeneously in Drosophila melanogaster S2 cells but shifted exclusively to the nucleus, if Usp is present.

Asunto(s)

Drosophila melanogaster/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo , Proteína Exportina 1

RESUMEN

Cathepsins are expressed in antigen-presenting cells (APC). These cathepsins are known to regulate antigen processing and degradation of the invariant chain (Ii) into the class II-associated Ii peptide (CLIP), which occupies the peptide-binding groove of the major histocompatibility complex (MHC) class II molecule. Previous studies have identified the serine carboxypeptidase cathepsin A (CatA) in various tissues and cells; however, it is not clear whether CatA is also expressed in primary human APC. We demonstrate the expression of CatA in B lymphoblastoid cells (BLC), primary human B cells, both subsets of myeloid dendritic cells (mDC1 and mDC2), as well as in plasmacytoid DC. PMSF or lactacystin-mediated inhibition of serine proteases in BLC-derived lysosomal proteases resulted in the inhibition of amino acid release from the C-terminal end of two model peptides. This inhibition did not occur by using a proline rich peptide. Our data suggest that CatA is involved in the C-terminal fine-tuning of antigenic T cell epitopes in human APC.

Asunto(s)

Células Presentadoras de Antígenos , Catepsina A/metabolismo , Secuencia de Aminoácidos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/química , Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología

RESUMEN

Several reviews devoted to various aspects of ecdysone research have been published during the last few years. Therefore, this article concentrates mainly on the considerable progress in ecdysone research observed recently, and will cover the results obtained during the last 2 years. The main emphasis is put on the molecular mode of ecdysteroid receptor-mediated hormone action. Two examples of interaction with other hormonal signalling pathways are described, namely crosstalk with juvenile hormone and insulin. Some selected, recently investigated examples of the multitude of hormonal responses are described. Finally, ecological aspects and some practical applications are discussed.

Asunto(s)

Ecdisteroides/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal , Animales , Artrópodos/crecimiento & desarrollo , Artrópodos/metabolismo , Ecdisona/metabolismo , Modelos Biológicos , Sesquiterpenos/metabolismo

RESUMEN

CHO-K1 cells are routinely used for characterization of ecdysone receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the receptor protein showing a minimum of receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1.

Asunto(s)

Ciclo Celular/fisiología , Receptores de Esteroides/metabolismo , Animales , Células CHO , Ciclo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Ciclina D1/metabolismo , Ecdisona/farmacología , Regulación de la Expresión Génica

RESUMEN

The glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase widely expressed in mammalian tissues. Initially identified by its ability to modulate glycogen synthesis, GSK-3 turned out to be a multifunctional enzyme, able to phosphorylate many proteins, including members of the steroid receptor superfamily. Although GSK-3 was shown to phosphorylate the androgen receptor (AR), its effects on AR transcriptional activity remain controversial. Analysis of short hairpin RNA (shRNA)-mediated downmodulation of GSK-3 proteins in prostate cancer cells showed a reduction in AR transcriptional activity and AR protein levels. Pharmacological GSK-3 inhibitors such as the maleimide SB216763 or the aminopyrazole GSK inhibitor XIII inhibited AR-dependent reporter gene activity and AR expression in vitro. Analysis of androgen-induced nuclear translocation of the AR was performed in PC3 cells transfected with pAR-t1EosFP coding for EosAR, a green fluorescent AR fusion protein. When grown in presence of androgens, EosAR was predominantly nuclear. Incubation with SB216763 before and after androgen treatment almost completely reduced nuclear EosAR. In contrast, the thiazole-containing urea compound AR-A014418 increased rather than decreased AR-expression/function. Although not all GSK inhibitors affected AR-stability/function, our observations suggest a potential new therapeutic application for some of these compounds in prostate cancer.

Asunto(s)

Andrógenos/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias de la Próstata/metabolismo , Transporte Activo de Núcleo Celular , Antineoplásicos/farmacología , Proliferación Celular , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Indoles/farmacología , Masculino , Maleimidas/farmacología , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Transcripción Genética

RESUMEN

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.

Asunto(s)

Luz , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Mutantes/metabolismo , Absorción , Biomarcadores/química , Biomarcadores/metabolismo , Dimerización , Fluorescencia , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación , Estructura Cuaternaria de Proteína , Factores de Tiempo , Proteína Fluorescente Roja

RESUMEN

Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , ADN/metabolismo , Dimerización , Proteínas de Drosophila , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptor alfa X Retinoide/metabolismo , Transcripción Genética

RESUMEN

BACKGROUND: Deregulation of the canonical Wnt/beta-catenin-pathway is known to play an important role in the progression of various tumour cell types including prostate cancer (PCa). Recently, the tumour-suppressor p53 was shown to down-regulate beta-catenin-signalling in colon cancer. As p53 is frequently mutated in late stage PCa we investigated the effect of wild-type p53 (p53wt) as well as p53-mutants on beta-catenin-signalling in PCa-cell lines. METHODS: The effects of p53wt and p53-mutants on Wnt/beta-catenin-signalling were studied using reporter gene assays. Expression of beta-catenin levels was monitored by Western blotting. RESULTS: Overexpression of p53wt as well as p53(249Ser) (a structural mutant) and p53(273His) (a DNA-contact-mutant) almost completely inhibited beta-catenin-mediated transcriptional activity of the T-cell factor (TCF) whereas p53(175His), a structural mutant, and a p53-mutant with a C-terminal deletion in the tetramerization domain (Deltap53) were unable to do so. Co-transfection experiments with p53wt and a dominant negative p53-mutant reversed the down-regulation of TCF-signalling, while Deltap53 was unable to interfere with p53wt-function. Down-regulation of TCF-signalling by p53wt and p53(273His) was accompanied by a reduction in beta-catenin protein level. CONCLUSIONS: p53wt, p53(273His)- and p53(249Ser)-mutants are able to down-regulate beta-catenin-signalling in PCa-cells probably via degradation of beta-catenin. The degradation of beta-catenin in PCa by p53 is not linked to transcriptional activity of p53. So far the mechanism how p53 interferes with beta-catenin-signalling is unknown. For the first time we provide experimental evidence that the C-terminus of p53 plays an important role in the down-regulation of beta-catenin-mediated TCF-signalling in PCa-cell lines possibly via p53 transrepressional function.

Asunto(s)

Mutación , Neoplasias de la Próstata/metabolismo , Factores de Transcripción TCF/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismo , Western Blotting , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Genes p53 , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Transducción de Señal , Factores de Transcripción TCF/genética , Proteína 2 Similar al Factor de Transcripción 7 , Activación Transcripcional , Transfección , beta Catenina/agonistas

RESUMEN

In the absence of hormone the ecdysteroid receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5-10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcR(M504R) is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcR(M504A) indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcR(M504R) is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slightly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.

Asunto(s)

Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Ecdisterona/análogos & derivados , Receptores de Esteroides/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ecdisterona/metabolismo , Ligandos , Mutación , Estructura Terciaria de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección

RESUMEN

The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster , Metabolismo Energético/fisiología , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas de Drosophila , Ácidos Grasos Insaturados , Células HeLa , Humanos , Oligomicinas , Unión Proteica , Proteína Exportina 1

RESUMEN

The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals.

Asunto(s)

Quitina/análisis , Poríferos/química , Poríferos/ultraestructura , Animales , Microscopía Confocal , Microscopía Fluorescente , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Difracción de Rayos X

RESUMEN

Ecdysteroids coordinate development, reproduction and other essential biological processes in insects and other arthropods through the receptor which is a heterodimer of two members of the nuclear receptors superfamily, the ecdysteroid receptor (EcR) and the Ultraspiracle (Usp). Although the transcriptionally active EcR/Usp heterocomplex is believed to be the only functional form of the receptor, there are data indicating that EcR may be involved in the mediation of the non-genomic effects outside of the nucleus. Since the nucleocytoplasmic shuttling could be a key element determining participation of the single nuclear receptor molecule both in the genomic and non-genomic functions we have analyzed nuclear import and export properties of the EcR and Usp from Drosophila melanogaster. We show for the first time that both receptors exhibit differential distribution of the nuclear localization and nuclear export signals (NLSs and NESs). In particular, the Usp which exhibits exclusively nuclear localization in all cell types analyzed, contains apparently only NLS activity within the DNA-binding domain. In contrast, the three known EcR isoforms (A, B1 and B2) are mosaics of elements which can potentially mediate their nucleocytoplasmic shuttling. We have found two active NESs in ligand binding domain and NLS activity within the DNA-binding domain of all isoforms. Simultaneously we demonstrate that B1 and A isoforms possess an additional NLS activity localized in AB regions. We speculate that this characteristic, along with the previously reported structural pliability of the EcR molecule, allows the single receptor to evoke many different genomic as well as non-genomic ecdysteroid-dependent responses.

Asunto(s)

Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Señales de Exportación Nuclear/genética , Señales de Exportación Nuclear/fisiología , Señales de Localización Nuclear/metabolismo , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Factores de Transcripción/genética

RESUMEN

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.

Asunto(s)

Núcleo Celular/metabolismo , Receptores de Esteroides/metabolismo , Receptores X Retinoide/metabolismo , Animales , Proteínas Bacterianas/genética , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide/genética

RESUMEN

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.

Asunto(s)

Proteínas Luminiscentes/química , Proteínas Luminiscentes/efectos de la radiación , Animales , Antozoos/química , Antozoos/genética , Color , Genes Reporteros , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Fotoquímica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efectos de la radiación , Espectrometría de Fluorescencia , Rayos Ultravioleta

RESUMEN

We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4-10. Red fluorophore maturation reaches a level of 90% after approximately 12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS/PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture.

Asunto(s)

Cnidarios/química , Proteínas Luminiscentes/fisiología , Secuencia de Aminoácidos , Animales , Biopolímeros , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Proteínas Luminiscentes/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Proteína Fluorescente Roja

RESUMEN

An epithelial cell line from Chironomus tentans exhibits acetylcholinesterase activity (specific activity 0.05-0.2 nkat/mg protein), which rises 30- to 40-fold after addition of 10-6 M 20-OH-ecdysone. The first visible increase occurs after 4 days of incubation with hormone. The enzyme has an apparent K m of 2.3±0.2×10-4 M for acetylthiocholine iodide as substrate and is inhibited by eserine and BW284 C51 (50% inhibition at 5×10-7 M for both inhibitiors) as well as by high concentrations of substrate, but not by tetraisopropylpyrophosphamide. The sensitivity against inhibitors is the same in extracts from hormone-treated cells and from controls. The cholinesterase activity correlates with morphological changes (shape and cell arrangement) and is indepenent of neuronal differentiation. We therefore propose a function for this activity during morphogenesis.

RESUMEN

The planulae ofHydractinia, the metamorphosis of which normally is induced by certain bacteria, will undergo transformation into polyps also when exposed to a lithiumpulse. The optimal concentration and incubation period for rapid and complete transformation have been determined at 24 mM Li+ and 2 hrs respectively. 96 mM K+ applied for 2 hrs will also result in some induction. The possible mode of action exerted by the Li-ion as compared with induction caused by bacteria is discussed.