Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Steroid hormones interrelationships in the metabolic syndrome: an introduction to the ponderostat hypothesis

Sexual dimorphism in the metabolic syndrome. The clairvoyant early implication of sex hormones in the characterization of the metabolic syndrome (MS) was detected early, and in accordance with the well-known sex-related main patterns of fat deposition in obesity: gynoid and android. The differences point to a direct implication of androgens and estrogens in the development, properties and maintenance of obesity and, by extension, to the cumulus of diseases grouped in the MS. For a long time, the key issue of the MS, i.e. the metabolic event explaining (and justifying) most of the derangements of the MS, has been considered to be insulin resistance (...)

Estudio del desarrollo y conectividad de las neuronas vasotocinérgicas en el prosencéfalo de pollo

En este Trabajo de Fin de Grado se estudia el origen embrionario de las distintas poblaciones neuronales que forman la amígdala medial extendida. La amígdala es una estructura del cerebro anterior involucrada en otorgar un significado emocional a los estímulos ambientales y en el control de distintos aspectos del comportamiento social (p.ej. comportamientos sexual, maternal, agresivo y afiliativo). Ante dichos estímulos, la amígdala pone en marcha una serie de reacciones de carácter motor, autonómico y endocrino que constituyen la respuesta emocional. Algunos desórdenes de carácter neuropsiquiátrico en humanos están relacionados con una disfunción en el control de las emociones y del comportamiento social, y varios de ellos se asocian a alteraciones en el desarrollo de la amígdala. El objetivo del presente trabajo ha sido investigar el origen de las neuronas de la amígdala medial extendida en embriones de pollo (E15 y E18) mediante ensayos de inmunocitoquímica, técnica utilizada para localizar las células que contienen el neuropéptido vasotocina (AVT) y proteínas reguladoras del desarrollo (la producida a partir del gen Otp) para ayudar en la delimitación de los distintos dominios embrionarios del prosencéfalo y distintas subdivisiones de la amígdala extendida. Los resultados de estos ensayos se combinaron con ensayos de trazado de conexiones para analizar la conectividad de las neuronas vasotocinérgicas de esta estructura. Los resultados obtenidos sugieren que las neuronas AVT-positivas podrían derivar del dominio Supra-Opto-Paraventricular (SPV), y algunas poblaciones alcanzarían su posición definitiva dentro del propio dominio por migración radial, mientras que otras invadirían otros dominios cerebrales por migración tangencial. En conclusión, la investigación proporciona importantes datos que clarifican aspectos relevantes del desarrollo y organización adulta de la amígdala extendida, y ayuda a establecer las bases para una mejor comprensión del control neural de las emociones y el comportamiento social en condiciones normales y patológicas.