The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

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.

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.

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

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.

Verifique sus conocimientos sobre medicina nuclear (2): tomografía por emisión de positrones (PET) y PET con tomografía computerizada (PET-TC)

La presente entrega de la serie de Nursing sobre las pruebas complementarias está dedicada a la tomografía por emisión de positrones o PET, acrónimo de positron emission tomography. La PET es una técnica de diagnóstico por la imagen de medicina nuclear en la cual se administra al paciente un radiofármaco emisor de positrones. Este radiofármaco se incorpora a los tejidos adecuados siguiendo una vía metabólica determinada. La radiactividad emitida por esos tejidos del paciente es detectable por los equipos PET y se obtienen imágenes que proporcionan una información funcional in vivo. El radiofármaco PET más habitual es un análogo de la glucosa que se llama F-18-fluordesoxiglucosa, conocido como FDG, el cual permite estudiar la actividad metabólica. La incorporación de la tomografía computarizada (TC) en el mismo equipo híbrido PET-TC permite obtener además la información anatómica del paciente. En el presente artículo se describen los fundamentos físicos y fisiológicos básicos de las exploraciones PET-TC con FDG en oncología, así como los procedimientos de enfermería necesarios para el cuidado del paciente y la correcta obtención de las imágenes.

Verifique sus conocimientos sobre medicina nuclear (2): tomografía por emisión de positrones (PET) y PET con tomografía computerizada (PET-TC)

La presente entrega de la serie de Nursing sobre las pruebas complementarias está dedicada a la tomografía por emisión de positrones o PET, acrónimo de positron emission tomography. La PET es una técnica de diagnóstico por la imagen de medicina nuclear en la cual se administra al paciente un radiofármaco emisor de positrones. Este radiofármaco se incorpora a los tejidos adecuados siguiendo una vía metabólica determinada. La radiactividad emitida por esos tejidos del paciente es detectable por los equipos PET y se obtienen imágenes que proporcionan una información funcional in vivo. El radiofármaco PET más habitual es un análogo de la glucosa que se llama F-18-fluordesoxiglucosa, conocido como FDG, el cual permite estudiar la actividad metabólica. La incorporación de la tomografía computarizada (TC) en el mismo equipo híbrido PET-TC permite obtener además la información anatómica del paciente. En el presente artículo se describen los fundamentos físicos y fisiológicos básicos de las exploraciones PET-TC con FDG en oncología, así como los procedimientos de enfermería necesarios para el cuidado del paciente y la correcta obtención de las imágenes.

Síntesi de la molècula ML-10 i del seu precursor immediat per poder obtenir 18F-ML-10, d'ús en tècniques PET

El 18F-ML-10 és un radiofàrmac utilitzat com a traçador de cèl·lules apoptòtiques mitjançant la tècnica del PET (Positron Emission Tomography). En el present treball de recerca s’ha dut a terme la síntesi de dos precursors del 18F-ML-10, i del producte final no marcat isotòpicament, el ML-10. S’ha plantejat una síntesi convergent per l’obtenció del compost final 3, partint del malonat de di-tert-butil i l’1,5-pentandiol. Tant el precursor tosilat 1 com el mesilat 2 s’han sintetitzat en 3 etapes amb un 29% i un 36% de rendiment, respectivament. El compost 3, utilitzat com a referència en la síntesi del 18F-ML-10, s’ha sintetitzat en 2 etapes a partir del precursor 2 amb un rendiment total de la síntesi del 31%. El procés de radiomarcació s’està portant a terme a l’Institut d’Alta Tecnologia – Parc de Recerca Biomèdica de Barcelona (IAT–PRBB), on posteriorment es faran estudis neurològics i oncològics on el seguiment de processos apoptòtics és clau.

New Radicals and new applications of Dynamic Nuclear Polarization of biological systems

Dynamic Nuclear Polarization (DNP) is an emerging technique that could revolutionize the NMR study of small molecules at very low concentrations by the increase in sensitivity that results from transfer of polarization between electronic and nuclear spins. Although the underlying physics has been known for a long time, in the last few years there has been a lot of excitement on the chemistry and biology NMR community caused by the demonstration that the highly polarized nuclei that are prepared in solid state at very low temperatures (1-2 K) could be rapidly transferred to liquid samples at room temperature and studied in solution by conventional NMR techniques. In favorable cases several order of magnitude increases in sensitivity have been achieved. The technique is now mature enough that a commercial instrument is available. The efficiency of DNP depends on two crucial aspects: i) the efficiency of the nuclear polarization process and ii) the efficiency of the transfer from the initial solid state to the fluid state in which NMR is measured. The preferred areas of application (iii) will be dictated by situations in which the low concentration of the sample or its intrinsic low receptivity are the limiting factors .

Polychlorinated trityl radicals for dynamic nuclear polarization: the role of chlorine nuclei

Polychlorinated trityl radicals bearing carboxylate substituents are water soluble persistent radicals that can be used for dynamic nuclear polarization. In contrast to other trityl radicals, the polarization mechanism differs from the classical solid effect. DFT calculations performed to rationalize this behaviour support the hypothesis that polarization is transferred from the unpaired electron to chlorine nuclei and from these to carbon by spin diffusion. The marked differences observed between neutral and anionic forms of the radical will be discussed.

Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta

Background There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression. Methodology/Principal Findings Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1β is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1β increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1β-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1β increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor α (ERRα). Conclusions/Significance Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1β in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

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.

Verifique sus conocimientos sobre medicina nuclear (1): gammagrafía

La presente entrega de la serie de Nursing sobre pruebas complementarias está dedicada a la gammagrafía. La gammagrafía es una técnica de diagnóstico por la imagen de medicina nuclear. Las exploraciones de medicina nuclear se pueden clasificar en medicina nuclear convencional (gammagrafía) y medicina nuclear por tomografía por emisión de positrones (PET), que se estudiará en la siguiente entrega de Nursing sobre pruebas complementarias. La gammagrafía es una técnica diagnóstica que utiliza sustancias radiactivas (isótopos) para estudiar la anatomía y el funcionalismo de diferentes órganos y tejidos del cuerpo. La gran ventaja de esta modalidad diagnóstica es su carácter funcional y su capacidad para evidenciar procesos pre-anatómicos de desarrollo patológico o anómalo que, junto con su elevada sensibilidad, permite diagnosticar alteraciones en fases muy precoces para poder ser tratadas. En el presente artículo se exponen las pruebas diagnósticas de gammagrafía más habituales, teniendo en cuenta que también existe la posibilidad de tratamientos terapéuticos que no son objeto de este trabajo. La enfermera, además del cuidado del paciente durante la preparación y después de la técnica, puede resolver las inquietudes relacionadas con las exploraciones gammagráficas, que normalmente tienen que ver con las características de cada prueba, la duración y el grado de molestia.

Verifique sus conocimientos sobre medicina nuclear (1): gammagrafía

La presente entrega de la serie de Nursing sobre pruebas complementarias está dedicada a la gammagrafía. La gammagrafía es una técnica de diagnóstico por la imagen de medicina nuclear. Las exploraciones de medicina nuclear se pueden clasificar en medicina nuclear convencional (gammagrafía) y medicina nuclear por tomografía por emisión de positrones (PET), que se estudiará en la siguiente entrega de Nursing sobre pruebas complementarias. La gammagrafía es una técnica diagnóstica que utiliza sustancias radiactivas (isótopos) para estudiar la anatomía y el funcionalismo de diferentes órganos y tejidos del cuerpo. La gran ventaja de esta modalidad diagnóstica es su carácter funcional y su capacidad para evidenciar procesos pre-anatómicos de desarrollo patológico o anómalo que, junto con su elevada sensibilidad, permite diagnosticar alteraciones en fases muy precoces para poder ser tratadas. En el presente artículo se exponen las pruebas diagnósticas de gammagrafía más habituales, teniendo en cuenta que también existe la posibilidad de tratamientos terapéuticos que no son objeto de este trabajo. La enfermera, además del cuidado del paciente durante la preparación y después de la técnica, puede resolver las inquietudes relacionadas con las exploraciones gammagráficas, que normalmente tienen que ver con las características de cada prueba, la duración y el grado de molestia.