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1.
J Chem Inf Model ; 59(5): 2046-2062, 2019 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-30817167

RESUMEN

At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/química , Industria Farmacéutica , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Humanos , Modelos Moleculares , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Factores de Tiempo , Interfaz Usuario-Computador
2.
Arch Pharm Res ; 39(11): 1491-1502, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27699647

RESUMEN

Innate immunity constitutes the first line of defense against pathogenic and dangerous insults. However, it is a double-edged sword, as it functions in both clearance of infection and inflammatory damage. It is therefore important that innate immune responses are tightly controlled to prevent harmful excessive inflammation. Nuclear receptors (NRs) are a family of transcription factors that play critical roles in various physiological responses. Orphan NRs are a subset of NRs for which the ligands and functions are unclear. Accumulating evidence has revealed that orphan NRs play essential roles in innate immune responses to prevent pathogenic inflammatory responses and to enhance antimicrobial host defenses. In this review, we describe current knowledge on the roles and mechanisms of orphan NRs in the regulation of innate immune responses. Discovery of new functions of orphan NRs would facilitate development of novel preventive and therapeutic strategies against human inflammatory diseases.


Asunto(s)
Inmunidad Innata/fisiología , Receptores Nucleares Huérfanos/fisiología , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Ligandos , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/genética , Procesamiento Proteico-Postraduccional , Transducción de Señal
3.
BMC Genomics ; 17(1): 832, 2016 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-27782803

RESUMEN

BACKGROUND: NR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis. Nr2e1-null mice exhibit reduced cortical and limbic structures and pronounced retinal dystrophy. NR2E1 functions mainly as a repressor of gene transcription in association with the co-repressors atrophin-1, LSD1, HDAC and BCL11A. Recent evidence suggests that NR2E1 also acts as an activator of gene transcription. However, co-activator complexes that interact with NR2E1 have not yet been identified. In order to find potential novel co-regulators for NR2E1, we used a microarray assay for real-time analysis of co-regulator-nuclear receptor interaction (MARCoNI) that contains peptides representing interaction motifs from potential co-regulatory proteins, including known co-activator nuclear receptor box sequences (LxxLL motif). RESULTS: We found that NR2E1 binds strongly to an atrophin-1 peptide (Atro box) used as positive control and to 19 other peptides that constitute candidate NR2E1 partners. Two of these proteins, p300 and androgen receptor (AR), were further validated by reciprocal pull-down assays. The specificity of NR2E1 binding to peptides in the array was evaluated using two single amino acid variants, R274G and R276Q, which disrupted the majority of the binding interactions observed with wild-type NR2E1. The decreased binding affinity of these variants to co-regulators was further validated by pull-down assays using atrophin1 as bait. Despite the high conservation of arginine 274 in vertebrates, its reduced interactions with co-regulators were not significant in vivo as determined by retinal phenotype analysis in single-copy Nr2e1-null mice carrying the variant R274G. CONCLUSIONS: We showed that MARCoNI is a specific assay to test interactions of NR2E1 with candidate co-regulators. In this way, we unveiled 19 potential co-regulator partners for NR2E1, including eight co-activators. All the candidates here identified need to be further validated using in vitro and in vivo models. This assay was sensitive to point mutations in NR2E1 ligand binding domain making it useful to identify mutations and/or small molecules that alter binding of NR2E1 to protein partners.


Asunto(s)
Ligandos , Receptores Nucleares Huérfanos/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Secuencia de Aminoácidos , Animales , Proteínas Portadoras , Descubrimiento de Drogas , Variación Genética , Humanos , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo
4.
Diabetes ; 65(10): 2835-48, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27335230

RESUMEN

Estrogen-related receptor γ (ERRγ) is a major positive regulator of hepatic gluconeogenesis. Its transcriptional activity is suppressed by phosphorylation signaled by insulin in the fed state, but whether posttranslational modification alters its gluconeogenic activity in the fasted state is not known. Metabolically active hepatocytes direct a small amount of glucose into the hexosamine biosynthetic pathway, leading to protein O-GlcNAcylation. In this study, we demonstrate that ERRγ is O-GlcNAcylated by O-GlcNAc transferase in the fasted state. This stabilizes the protein by inhibiting proteasome-mediated protein degradation, increasing ERRγ recruitment to gluconeogenic gene promoters. Mass spectrometry identifies two serine residues (S317, S319) present in the ERRγ ligand-binding domain that are O-GlcNAcylated. Mutation of these residues destabilizes ERRγ protein and blocks the ability of ERRγ to induce gluconeogenesis in vivo. The impact of this pathway on gluconeogenesis in vivo was confirmed by the observation that decreasing the amount of O-GlcNAcylated ERRγ by overexpressing the deglycosylating enzyme O-GlcNAcase decreases ERRγ-dependent glucose production in fasted mice. We conclude that O-GlcNAcylation of ERRγ serves as a major signal to promote hepatic gluconeogenesis.


Asunto(s)
Gluconeogénesis/fisiología , Hígado/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Células Cultivadas , Gluconeogénesis/genética , Glicosilación , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/genética , Procesamiento Proteico-Postraduccional/fisiología , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Serina/química , Serina/metabolismo
5.
J Steroid Biochem Mol Biol ; 157: 1-2, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26791250

RESUMEN

This Special Issue on the topic of "Orphan Nuclear Receptors" should help to cement the long held view that orphan members of the Nuclear Receptor superfamily play crucial roles in development, physiology and multiple pathologies and that some are attractive druggable targets. Focusing on selected orphans, this issue highlights recent developments in orphan receptor action and addresses questions about function, ligand recognition, strategies for drug development and applications for such drugs. This article is part of a Special Issue entitled "Orphan Nuclear Receptors".


Asunto(s)
Receptores Nucleares Huérfanos/fisiología , Animales , Colesterol/metabolismo , Humanos , Ligandos , Terapia Molecular Dirigida/métodos , Neoplasias/metabolismo , Receptores Nucleares Huérfanos/química
6.
J Steroid Biochem Mol Biol ; 157: 3-6, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25797032

RESUMEN

This review summarizes the birth of the field of nuclear receptors, from Jensen's discovery of estrogen receptor alpha, Gustafsson's discovery of the three-domain structure of the glucocorticoid receptor, the discovery of the glucocorticoid response element and the first partial cloning of the glucocorticoid receptor. Furthermore the discovery of the novel receptors called orphan receptors is described.


Asunto(s)
Bioquímica/historia , Receptores Nucleares Huérfanos/historia , Animales , Clonación Molecular , ADN/historia , ADN/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/fisiología , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta
7.
J Steroid Biochem Mol Biol ; 157: 27-40, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26159912

RESUMEN

Nuclear receptors are defined as a family of ligand regulated transcription factors [1-6]. While this definition reflects that ligand binding is a key property of nuclear receptors, it is still a heated subject of debate if all the nuclear receptors (48 human members) can bind ligands (ligands referred here to both physiological and synthetic ligands). Recent studies in nuclear receptor structure biology and pharmacology have undoubtedly increased our knowledge of nuclear receptor functions and their regulation. As a result, they point to new avenues for the discovery and development of nuclear receptor regulators, including nuclear receptor ligands. Here we review the recent literature on orphan nuclear receptor structural analysis and ligand identification, particularly on the orphan nuclear receptors that do not heterodimerize with retinoid X receptors, which we term as non-X orphan receptors. We also propose a speculative "retinoid hypothesis" for a subset of non-X orphan nuclear receptors, which we hope to help shed light on orphan nuclear receptor biology and drug discovery. This article is part of a Special Issue entitled 'Orphan Nuclear Receptors'.


Asunto(s)
Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Retinoides/metabolismo , Animales , Sitios de Unión , Factor de Transcripción COUP II/química , Factor de Transcripción COUP II/metabolismo , Receptor Nuclear Huérfano DAX-1/química , Receptor Nuclear Huérfano DAX-1/metabolismo , Humanos , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/química , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Conformación Proteica , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Retinoides/química , Factor Esteroidogénico 1/química , Factor Esteroidogénico 1/metabolismo
8.
Artículo en Inglés | MEDLINE | ID: mdl-26342960

RESUMEN

Liver X Receptor (LXR) plays a pivotal role in metabolic regulation in mammals, but little is known about its function in fish. In this study, two lxra isoforms, namely lxra1 and lxra2, were isolated. Their molecular characterization, tissues distribution and transcriptional regulation by insulin in vivo and in vitro were determined. lxrα1 and lxrα2 cDNA covered 2775bp and 3093bp, encoding 446 and 515 amino acid residues, respectively. The protein sequence of yellow catfish lxra included characteristic feature of mammalian lxrs, including the DNA binding (DBD) (containing P-box), ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, and D (hinge) region. Phylogenetic analysis revealed that yellow catfish lxra grouped with lxra of zebrafish but was distant from those of medaka and stickleback. lxrß clades was absent in teleosts in phylogenetic tree, proving gene loss of lxrß in teleosts during evolution. The two lxra isoforms (lxra1 and lxra2) mRNAs were ubiquitously expressed in 11 tested tissues. Compared to lxra2, lxra1 mRNA expression was predominant in all tested tissues. The expression of lxrα1 was the highest in testis, then in liver, and the lowest in other tissues. lxrα2 expression was the highest in liver, then in testis, and the lowest in ovary. Insulin significantly stimulated the mRNA expression of lxra1 in vitro and in vivo, while the expression of lxra2 remained unchanged after insulin treatment. The present study serves to increase our understanding into the function of lxra in fish.


Asunto(s)
Bagres/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Receptores Nucleares Huérfanos/genética , Transcripción Genética/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos/química , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo
9.
Biochem Biophys Res Commun ; 469(3): 573-9, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26692490

RESUMEN

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.


Asunto(s)
Regulación de la Expresión Génica/genética , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Activación Transcripcional/genética , Sitios de Unión , Células Hep G2 , Factor Nuclear 4 del Hepatocito/química , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos/química , Unión Proteica
10.
Lipids ; 50(12): 1195-207, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26494560

RESUMEN

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or ß. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/agonistas , Anticolesterolemiantes/farmacología , Antineoplásicos Fitogénicos/farmacología , Colesterol/metabolismo , Glioblastoma/tratamiento farmacológico , Receptores Nucleares Huérfanos/agonistas , Estilbenos/farmacología , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Hidroxicolesteroles/metabolismo , Hidroxilación , Cinética , Receptores X del Hígado , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo
11.
Crit Rev Biochem Mol Biol ; 50(5): 380-92, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26383043

RESUMEN

Health during aging can be improved by genetic, dietary and pharmacological interventions. Many of these increase resistance to various stressors, including xenobiotics. Up-regulation of xenobiotic detoxification genes is a transcriptomic signature shared by long-lived nematodes, flies and mice, suggesting that protection of cells from toxicity of xenobiotics may contribute to longevity. Expression of genes involved in xenobiotic detoxification is controlled by evolutionarily conserved transcriptional regulators. Three closely related subgroups of nuclear hormone receptors (NHRs) have a major role, and these include DAF-12 and NHR-8 in C. elegans, DHR96 in Drosophila and FXR, LXRs, PXR, CAR and VDR in mammals. In the invertebrates, these NHRs have been experimentally demonstrated to play a role in extension of lifespan by genetic and environmental interventions. NHRs represent critical hubs in that they regulate detoxification enzymes with broad substrate specificities, metabolizing both endo- and xeno-biotics. They also modulate homeostasis of steroid hormones and other endogenous cholesterol derivatives and lipid metabolism, and these roles, as well as xenobiotic detoxification, may contribute to the effects of NHRs on lifespan and health during aging, an issue that is being increasingly addressed in C. elegans and Drosophila. Disentangling the contribution of these processes to longevity will require more precise understanding of the molecular mechanisms by which each is effected, including identification of ligands and co-regulators of NHRs, patterns of tissue-specificity and mechanisms of interaction between tissues. The roles of vertebrate NHRs in determination of health during aging and lifespan have yet to be investigated.


Asunto(s)
Envejecimiento/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Receptores Citoplasmáticos y Nucleares/metabolismo , Xenobióticos/toxicidad , Animales , Biotransformación , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Humanos , Ligandos , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Conformación Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Esteroles/metabolismo , Toxicocinética , Xenobióticos/metabolismo , Xenobióticos/farmacocinética
12.
Int J Comput Biol Drug Des ; 8(1): 75-86, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25869321

RESUMEN

LXR (encoded by NR1H2 and 3) and FXR (known as bile acid receptor) encoded by NR1H4 (nuclear receptor subfamily 1, group H and member 4) are nuclear receptors in humans and are important regulators of bile acid production, cholesterol, fatty acid and glucose homeostasis hence responsible for liver detoxification. Several strategies for drug design with numerous ligands for this target have failed owing to the inability of the ligand to access the target/receptor or their early metabolisation. In this work, we have evaluated FXR and LXR structure bound with agonist and compared the binding energy affinity of active ligands present in live green-real veggies with reference drugs (ligands) present in the market. A high throughput screening combined with molecular docking, absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions, log P values and percentage of human oral absorption value led to the identification of two compounds present in live green-real veggies with strong potential for liver detoxification.


Asunto(s)
Hígado/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Verduras/química , Simulación por Computador , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Inactivación Metabólica , Hígado/química , Hígado/metabolismo , Receptores X del Hígado , Simulación del Acoplamiento Molecular , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Extractos Vegetales/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo
13.
Mol Biosyst ; 11(5): 1241-50, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25734698

RESUMEN

Liver X receptor (LXR) α and ß are cholesterol sensors; they respond to excess cholesterol and stimulate reverse cholesterol transport. Activating LXRs represents a promising therapeutic option for dyslipidemia. However, activating LXRα may cause unwanted lipogenicity. A better anti-dyslipidemia strategy would be to develop selective LXRß agonists that do not activate LXRα. In this paper, a data set of 234 selective and non-selective LXRß agonists was collected from the literature. For the first time, we derived the classification models from the data set to predict selective LXRß agonists using multiple machine learning methods (naïve Bayesian (NB), Recursive Partitioning (RP), Support Vector Machine (SVM), and k-Nearest Neighbors (kNN) methods) with optimized property descriptors and structural fingerprints. The models were optimized from 324 multiple machine learning models, and most of the models showed high predictive abilities (overall predictive accuracies of >80%) for both training and test sets. The top 15 models were evaluated using an external test set of 76 compounds (all containing new scaffolds), and 10 of them displayed overall predictive accuracies exceeding 90%. The top models can be used for the virtual screening of selective LXRß agonists. The NB models can identify privileged and unprivileged fragments for selective LXRß agonists, and the fragments can be used to guide the design of new selective LXRß agonists.


Asunto(s)
Descubrimiento de Drogas , Aprendizaje Automático , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/química , Algoritmos , Descubrimiento de Drogas/métodos , Humanos , Receptores X del Hígado , Modelos Teóricos , Receptores Nucleares Huérfanos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados
14.
Hum Mutat ; 36(6): 599-610, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25703721

RESUMEN

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.


Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/genética , Dominios y Motivos de Interacción de Proteínas/genética , Multimerización de Proteína , Degeneración Retiniana/genética , Trastornos de la Visión/genética , Adolescente , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Línea Celular , Análisis Mutacional de ADN , Enfermedades Hereditarias del Ojo/diagnóstico , Proteínas del Ojo/metabolismo , Angiografía con Fluoresceína , Proteínas de Homeodominio/metabolismo , Humanos , Ligandos , Masculino , Modelos Moleculares , Mutación , Linaje , Unión Proteica , Conformación Proteica , Retina/metabolismo , Degeneración Retiniana/diagnóstico , Tomografía de Coherencia Óptica , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Trastornos de la Visión/diagnóstico
15.
J Steroid Biochem Mol Biol ; 149: 80-8, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25661920

RESUMEN

Liver X receptors (LXRs) monitor endogenous sterol levels to maintain whole-body cholesterol levels and regulate inflammatory responses. Recent studies have demonstrated that LXRs may inhibit cellular proliferation, but the underlying mechanism remains unclear. Cell cycle and apoptosis regulator 2 (CCAR2), previously known as DBC1/KIAA1967, is a transcriptional regulator that regulates cellular proliferation and energy metabolism by inhibiting sirtuin 1 (SIRT1) deacetylase. Based on the findings that CCAR2 regulates several nuclear receptors, including the estrogen receptors and androgen receptor, we aimed to identify the underlying mechanism of CCAR2 regulation of LXRα. We found that CCAR2 formed a complex with LXRα in a ligand-independent manner in HepG2 cells, and in vitro pull-down assays, it revealed a direct interaction between the amino terminus of CCAR2 and the AF-2 domain of LXRα. Thereby, CCAR2 attenuates the ligand-dependent transcriptional activation function of LXRα. RNA interference-mediated depletion of endogenous CCAR2 potentiated the expression of the LXRα target genes ATP-binding cassette transporter A1 and G1, and the abrogation of CCAR2 resulted in decreased cellular proliferation. Moreover, competitive immunoprecipitation studies revealed that the LXRα downregulation involves the inhibition of SIRT1-LXRα complex formation. Therefore, these results clearly indicate a novel mechanism in which CCAR2 may regulate the transcriptional activation function of LXRα due to its specific inhibition of SIRT1 and serve to regulate cellular proliferation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Mapas de Interacción de Proteínas , Sirtuina 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proliferación Celular , Células HEK293 , Células Hep G2 , Humanos , Receptores X del Hígado , Células MCF-7 , Receptores Nucleares Huérfanos/química , Dominios y Motivos de Interacción de Proteínas , Activación Transcripcional
16.
Genes Dev ; 29(4): 440-50, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25691470

RESUMEN

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX-Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.


Asunto(s)
Drosophila melanogaster/química , Modelos Moleculares , Receptores Nucleares Huérfanos/química , Receptores Citoplasmáticos y Nucleares/química , Animales , Cristalización , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Receptores Nucleares Huérfanos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo
17.
Steroids ; 96: 121-31, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25668616

RESUMEN

The individual chemical components of commercial extract of Gymnema sylvestre, a medicinal plant used in the traditional systems of the Indian medicine for its antidiabetic and hypolipidemic properties, were isolated and evaluated for their capability to act as modulators of nuclear and membrane receptors involved in glucose and lipid homeostasis. The study disclosed for the first time that individual gymnemic acids are potent and selective antagonists for the ß isoform of LXR. Indeed the above activity was shared by the most abundant aglycone gymnemagenin (10) whereas gymnestrogenin (11) was endowed with a dual LXRα/ß antagonistic profile. Deep pharmacological investigation demonstrated that gymnestrogenin, reducing the expression of SREBP1c and ABCA1 in vitro, is able to decrease lipid accumulation in HepG2 cells. The results of this study substantiate the use of G. sylvestre extract in LXR mediated dislypidemic diseases.


Asunto(s)
Descubrimiento de Drogas , Gymnema sylvestre/química , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Saponinas/química , Saponinas/farmacología , Triterpenos/química , Triterpenos/farmacología , Células Hep G2 , Humanos , Receptores X del Hígado , Modelos Moleculares , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/genética , Conformación Proteica , Activación Transcripcional/efectos de los fármacos
18.
J Microbiol Biotechnol ; 25(2): 247-54, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25223325

RESUMEN

In this presentation, I describe the expression and purification of the recombinant liver X receptor ß-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor ß-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor ß-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.


Asunto(s)
Escherichia coli/genética , Receptores Nucleares Huérfanos/química , Receptores Nucleares Huérfanos/metabolismo , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/metabolismo , Sitios de Unión , Western Blotting , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/microbiología , Ligandos , Receptores X del Hígado , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Receptores Nucleares Huérfanos/genética , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Receptor alfa X Retinoide/genética , Solubilidad , Factores de Transcripción
19.
Trends Biochem Sci ; 40(1): 25-35, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25499868

RESUMEN

Nuclear receptors (NRs) are key players in the regulation of gene expression, coordinating protein assemblies upon their surfaces. NRs are regulated by ligand binding, which remodels the interaction surfaces and subsequently influences macromolecular complex formation. Structural biology has been instrumental in the discovery of some of these ligands, but there are still orphan NRs (ONRs) whose bona fide ligands have yet to be identified. Over the past decade, fundamental structural and functional breakthroughs have led to a deeper understanding of ONR actions and their multidomain organization. Here, we summarize the structural advances in ONRs with implications for the therapeutic treatment of diseases such as metabolic syndrome and cancer.


Asunto(s)
Proteínas de Unión al ADN/química , Receptores Nucleares Huérfanos/química , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Humanos , Ligandos , Receptores Nucleares Huérfanos/metabolismo , Conformación Proteica , Relación Estructura-Actividad
20.
Mol Vis ; 20: 724-31, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24891813

RESUMEN

PURPOSE: Various autosomal recessive retinal dystrophies are reported to be associated with mutations in nuclear receptor subfamily 2, group E, member 3 (NR2E3, also called PNR) gene. The present study proposed to understand the clinical and genetic characteristics of the family of a patient with an ocular phenotype consistent with Goldmann-Favre syndrome (GFS) and vasoproliferative tumors of the retina (VPTRs). METHODS: Twelve family members of the proband from three generations underwent complete ophthalmic examination, including best-corrected visual acuity with Snellen optotypes, tonometry, biomicroscopic examination, indirect ophthalmoscopy after pupillary dilatation, computerized perimetry, optical coherence tomography, fundus photography, intravenous fluorescein angiography, and electroretinography (ERG). All the study subjects underwent genetic analysis of the entire coding region of the NR2E3 gene with the bidirectional DNA sequencing approach. Hundred healthy individuals were screened for the variant. RESULTS: The phenotype of the proband had features of GFS with VPTRs. The tumors showed complete resolution with cryotherapy and transpupillary thermotherapy (TTT). Sequencing of the entire coding region of the NR2E3 gene in the proband revealed a novel homozygous c.1117 A>G variant that led to the amino acid change from aspartic acid to glycine at position 406 (p.D406G). This change was present in the homozygous state in affected family members and in the heterozygous state in unaffected family members, and was undetectable in the control subjects. The identified novel p.D406G homozygous mutation was at an evolutionarily highly conserved region and may possibly affect the protein function (Sorting Intolerant From Tolerant [SIFT] score = 0.00). CONCLUSIONS: Patients with GFS may present with retinal VPTRs that respond to therapy with cryotherapy and TTT. Molecular genetic studies helped to identify a novel p.D406G mutation in the affected members, which will aid in confirming the diagnosis, for genetic counseling of family members and potentially provide some form of therapy for the affected patients.


Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación/genética , Receptores Nucleares Huérfanos/genética , Degeneración Retiniana/genética , Neoplasias de la Retina/irrigación sanguínea , Neoplasias de la Retina/genética , Trastornos de la Visión/genética , Adulto , Secuencia de Aminoácidos , Secuencia Conservada , Electrorretinografía , Evolución Molecular , Enfermedades Hereditarias del Ojo/fisiopatología , Familia , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Humanos , Masculino , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos/química , Linaje , Degeneración Retiniana/fisiopatología , Trastornos de la Visión/fisiopatología
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