RESUMEN
A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.
Asunto(s)
Técnicas de Química Analítica/métodos , Monitoreo del Ambiente/métodos , Receptor alfa de Estrógeno/química , Estrógenos/análisis , Contaminantes Químicos del Agua/análisis , Receptor alfa de Estrógeno/aislamiento & purificación , Colorantes Fluorescentes/química , Humanos , Ligandos , Unión Proteica , Coloración y EtiquetadoRESUMEN
A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Disruptores Endocrinos/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Sitios de Unión , Dimetilsulfóxido/química , Estradiol/química , Humanos , Interferometría/instrumentación , Interferometría/métodos , Ligandos , Modelos Moleculares , Ovalbúmina/química , Péptidos/química , Polietilenglicoles/química , Valor Predictivo de las Pruebas , Conformación Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis Espectral/instrumentación , Análisis Espectral/métodos , Propiedades de Superficie , Tamoxifeno/química , Factores de TiempoRESUMEN
A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.
Asunto(s)
Discos Compactos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Fosfopéptidos/análisis , Fosfopéptidos/química , Proteómica/instrumentación , Proteómica/métodos , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Datos de Secuencia Molecular , Fosfopéptidos/metabolismo , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The structures of the liver X receptor LXRbeta (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 A, respectively. Together with its isoform LXRalpha (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRbeta structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed "agonist conformation" of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/química , Alanina/química , Sitios de Unión , Colesterol/metabolismo , Proteínas de Unión al ADN , Dimerización , Electrones , Escherichia coli/metabolismo , Histidina/química , Humanos , Ligandos , Receptores X del Hígado , Modelos Químicos , Modelos Moleculares , Modelos Estadísticos , Receptores Nucleares Huérfanos , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Rayos XRESUMEN
Here we describe the three-dimensional crystal structures of human glucocorticoid receptor ligand-binding domain (GR-LBD) in complex with the antagonist RU-486 at 2.3 A resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 A. The RU-486 structure was solved in several different crystal forms, two with helix 12 intact (GR1 and GR3) and one with a protease-digested C terminus (GR2). In GR1, part of helix 12 is in a position that covers the co-activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys-736 residues in the two molecules. All three RU-486 GR-LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.
Asunto(s)
Dexametasona/química , Mifepristona/química , Receptores de Glucocorticoides/química , Animales , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Cisteína , Dexametasona/farmacología , Dimerización , Mifepristona/farmacología , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Spodoptera , TransfecciónRESUMEN
Un péptido sintético derivado de una región conservada de la glicoproteína de transmembrana gp41 compuesto por 12 aminoácidos fue evaluado abtígeno de fase sólida en un enzimoinmunoensayo. Se analizaron 3 diferentes paneles de suero de Suecia, la Argentina y Tanzania. Se encontró una especificidad del 97.7% y 97.2% para los sueros suecos y argentinos respectivamente, siendo la sensibilidad del 100% para ambos paneles. Para el panel africano la especificidad fue del 90.5% y la sensibilidad del 96.0%. Los resultados indican que este péptido es altamente reactivo con sueros positivos para HIVl y puede ser útil en ensayos de inmunodiagnóstico
Asunto(s)
Humanos , Aminoácidos/metabolismo , VIH-1/metabolismo , Péptidos/metabolismo , Síndrome de Inmunodeficiencia Adquirida/inmunología , Aminoácidos , Argentina , Ensayo de Inmunoadsorción Enzimática , VIH-1/inmunología , Péptidos , Suecia , TanzaníaRESUMEN
Un péptido sintético derivado de una región conservada de la glicoproteína de transmembrana gp41 compuesto por 12 aminoácidos fue evaluado abtígeno de fase sólida en un enzimoinmunoensayo. Se analizaron 3 diferentes paneles de suero de Suecia, la Argentina y Tanzania. Se encontró una especificidad del 97.7% y 97.2% para los sueros suecos y argentinos respectivamente, siendo la sensibilidad del 100% para ambos paneles. Para el panel africano la especificidad fue del 90.5% y la sensibilidad del 96.0%. Los resultados indican que este péptido es altamente reactivo con sueros positivos para HIVl y puede ser útil en ensayos de inmunodiagnóstico (AU)