RESUMO
Although somatic cell reprogramming to generate inducible pluripotent stem cells (iPSCs) is associated with profound epigenetic changes, the roles and mechanisms of epigenetic factors in this process remain poorly understood. Here, we identify Jmjd3 as a potent negative regulator of reprogramming. Jmjd3-deficient MEFs produced significantly more iPSC colonies than did wild-type cells, whereas ectopic expression of Jmjd3 markedly inhibited reprogramming. We show that the inhibitory effects of Jmjd3 are produced through both histone demethylase-dependent and -independent pathways. The latter pathway involves Jmjd3 targeting of PHF20 for ubiquitination and degradation via recruitment of an E3 ligase, Trim26. Importantly, PHF20-deficient MEFs could not be converted to fully reprogrammed iPSCs, even with knockdown of Jmjd3, Ink4a, or p21, indicating that PHF20 is required for reprogramming. Our findings demonstrate, to the best of our knowledge, a previously unrecognized role of Jmjd3 in cellular reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls this process.
Assuntos
Reprogramação Celular , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Cinética , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Regulação para CimaRESUMO
Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRß reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRß resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ2 mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially "rescued" the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/ß-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRß K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, ß-catenin, LEF1, and CCND1. Additionally, the TRß K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation.
Assuntos
Adipócitos/citologia , Diferenciação Celular , Proliferação de Células , Receptores dos Hormônios Tireóideos/metabolismo , Sumoilação , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Mutação , PPAR gama/genética , PPAR gama/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Receptores dos Hormônios Tireóideos/genéticaRESUMO
Many pathogens colonize different anatomical sites, but the selective pressures contributing to survival in the diverse niches are poorly understood. Group A Streptococcus (GAS) is a human-adapted bacterium that causes a range of infections. Much effort has been expended to dissect the molecular basis of invasive (sterile-site) infections, but little is known about the genomes of strains causing pharyngitis (streptococcal "sore throat"). Additionally, there is essentially nothing known about the genetic relationships between populations of invasive and pharyngitis strains. In particular, it is unclear if invasive strains represent a distinct genetic subpopulation of strains that cause pharyngitis. We compared the genomes of 86 serotype M3 GAS pharyngitis strains with those of 215 invasive M3 strains from the same geographical location. The pharyngitis and invasive groups were highly related to each other and had virtually identical phylogenetic structures, indicating they belong to the same genetic pool. Despite the overall high degree of genetic similarity, we discovered that strains from different host environments (i.e., throat, normally sterile sites) have distinct patterns of diversifying selection at the nucleotide level. In particular, the pattern of polymorphisms in the hyaluronic acid capsule synthesis operon was especially different between the two strain populations. This finding was mirrored by data obtained from full-genome analysis of strains sequentially cultured from nonhuman primates. Our results answer the long-standing question of the genetic relationship between GAS pharyngitis and invasive strains. The data provide previously undescribed information about the evolutionary history of pathogenic microbes that cause disease in different anatomical sites.
Assuntos
Evolução Molecular , Genoma Bacteriano/fisiologia , Faringite/genética , Filogenia , Infecções Estreptocócicas/genética , Streptococcus pyogenes/genética , Animais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , PrimatasRESUMO
The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.
Assuntos
Hipoglicemiantes/farmacologia , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Aumento de Peso , Células 3T3-L1 , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacocinética , Ligantes , Camundongos , Células NIH 3T3 , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Estrutura Secundária de Proteína , Tiazolidinedionas/química , Tiazolidinedionas/farmacocinética , Células U937RESUMO
UNLABELLED: Nuclear receptors (NRs) play crucial roles in the regulation of hepatic cholesterol synthesis, metabolism, and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured murine cholangiocytes and found that these cells express a specific subset of NRs, including liver X receptor (LXR) ß and peroxisome proliferator-activated receptor (PPAR) δ. Activation of LXRß and/or PPARδ in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPARδ induces Niemann-Pick C1-like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane via specific interaction with a peroxisome proliferator-activated response element (PPRE) within the NPC1L1 promoter. CONCLUSION: We propose that (1) LXRß and PPARδ coordinate NPC1L1/ABCA1-dependent vectorial cholesterol flux from bile through cholangiocytes and (2) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, two serious health concerns for humans.
Assuntos
Colesterol/metabolismo , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Ductos Biliares/citologia , Ductos Biliares/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Homeostase/genética , Receptores X do Fígado , Proteínas de Membrana Transportadoras/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptor de Pregnano X , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mammalian genomes are organized into multiple layers of higher-order chromatin structure, and in this organization chromatin looping is a striking and crucial feature that brings together distal genomic loci into close spatial proximity. Such three-dimensional organization of chromatin has been suggested to be functionally important in gene regulation. Many important questions need to be addressed, such as what types of nuclear proteins are responsible for folding chromatin into loops, whether there are any genomic marks that serve as the core sites of chromatin folding events, how distal genomic sites are brought together, and what are the biological consequences for interactions between distal genomic loci. In order to address these fundamental questions, it is essential to devise and employ methods that can capture higher-order structures formed by specific nuclear proteins at high resolution. In this article, in order to describe methods of analyzing protein-mediated chromatin interactions, we will use as an example a global genome-organizer protein, SATB1, which mediates chromatin looping.
Assuntos
Cromatina/genética , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Mapeamento Cromossômico/métodos , Reagentes de Ligações Cruzadas/química , Formaldeído/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the ß-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2', H3, and the ß-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators.
Assuntos
Luteolina/farmacologia , PPAR gama/agonistas , Células 3T3-L1 , Animais , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Luteolina/química , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ácido Mirístico/química , PPAR gama/química , PPAR gama/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Tiazolidinedionas/antagonistas & inibidores , Tiazolidinedionas/farmacologiaRESUMO
Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicates that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.
Assuntos
Proteínas de Ligação a Ácido Graxo , Ligantes , Estrutura Terciária de Proteína , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Chlorocebus aethiops , Cromanos/química , Cromanos/metabolismo , Cristalografia por Raios X , Dimerização , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Tiazolidinedionas/química , Tiazolidinedionas/metabolismo , TroglitazonaRESUMO
Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, including reductions in serum lipids, according to the current models these hormones act independently in vivo. In this study, we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRß-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRß. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRß1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WT and Fgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are maintained in the Fgf21(-/-) background and we observe no effect of the Fgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3 regulates the genes involved in classical hepatic metabolic responses independently of FGF21.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Fígado/metabolismo , Receptores beta dos Hormônios Tireóideos/fisiologia , Animais , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Elementos de Resposta , Tri-Iodotironina/farmacologiaRESUMO
Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor ß1 (TRß1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients.
Assuntos
Colesterol 7-alfa-Hidroxilase/genética , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/fisiologia , Adenoviridae/genética , Animais , Sequência de Bases , Colesterol 7-alfa-Hidroxilase/metabolismo , Indução Enzimática , Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Receptores beta dos Hormônios Tireóideos/genéticaRESUMO
Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRß binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRß appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRß binding sites immediately 5' and 3' of transcribed genes and TRß can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRß is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRß peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRß binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action.
Assuntos
Sítios de Ligação , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Genoma , Células Hep G2 , Humanos , Ligantes , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Ligação Proteica , RNA/química , Elementos de Resposta , Análise de Sequência de DNARESUMO
BACKGROUND: Method developers of plate-based ligand-binding assays (LBAs) often face challenges establishing selectivity, specificity and range of quantitation to meet the needs of a particular study. Case studies are presented to compare different ligand-binding immunoassay platforms (plate-based vs microfluidic system) in method development to support pharmacokinetic and pharmacodynamic studies. RESULTS: Studies highlight the challenges of plate-based LBAs to establish selectivity, specificity and range of quantitation as a result of nonspecific background signal, matrix interference, lack of linearity and drug interference. The fast assay kinetics of a microfluidic immunoassay system was shown to generally reduce nonspecific background and matrix effects, while increasing assay linear range and drug tolerance. CONCLUSION: The short incubation times with microfluidics can be beneficial for LBAs burdened by matrix effects and in these cases had superior assay performance compared with widely used immunoassay platforms in bioanalysis, for example, Meso Scale Discovery(®) and enzyme-linked immunosorbent assay.
Assuntos
Imunoensaio/instrumentação , Imunoensaio/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Biomarcadores/análise , Humanos , Ligantes , Sensibilidade e EspecificidadeRESUMO
Thyroid hormones (THs) are important in the development and maintenance of lipid and energy homeostasis. THs act through two closely related TH receptors (TRs α and ß), which are conditional transcription factors. Recently, TH analogues or thyromimetics with varying degrees of TR subtype and liver uptake selectivity have been developed. These compounds exert beneficial effects of TH excess states without many undesirable TR-dependent side effects. Several selective TR modulators (STRMs) showed exceptionally promising results in lowering serum cholesterol in preclinical animal models and human clinical studies. Moreover, some first generation STRMs elicit other potentially beneficial effects on obesity, glucose metabolism, and nonalcoholic fatty liver disease (NAFLD). While it was initially thought that STRMs would be an effective long-term therapy to combat elevated cholesterol, possibly in conjunction with another cholesterol-lowering therapy, the statins, three major first generation STRMs failed to progress beyond early phase III human trials. The aim of this review is to discuss how STRMs work, their actions in preclinical animal models and human clinical trials, why they did not progress beyond clinical trials as cholesterol-lowering therapeutics, whether selective TR modulation continues to hold promise for dyslipidemias, and whether members of this drug class could be applied to the treatment of other aspects of metabolic syndrome and human genetic disease.
Assuntos
Antitireóideos/química , Receptores dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/química , Animais , Química Farmacêutica/tendências , Colesterol/metabolismo , Ensaios Clínicos como Assunto , Desenho de Fármacos , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Lipídeos/química , Síndrome Metabólica/metabolismo , Hepatopatia Gordurosa não Alcoólica , Obesidade/metabolismo , Transdução de Sinais , Receptores beta dos Hormônios Tireóideos/metabolismoRESUMO
Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.
Assuntos
PPAR alfa/agonistas , PPAR alfa/química , Pirimidinas/química , Pirimidinas/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Gene expression is tightly regulated by transcription factors and cofactors that function by directly or indirectly interacting with DNA of the genome. Understanding how and where these proteins bind provides essential information to uncover genetic regulatory mechanisms. We have developed a new method to study DNA-protein interaction in vivo called DNA adenine methyltransferase (Dam)IP, which is based on fusing a protein of interest to a mutant form of Dam from Escherichia coli. We showed previously that DamIP can efficiently identify in vivo binding sites of Dam-tethered human estrogen receptor (hER)α. In current study, we present the cistrome of hERα determined by DamIP and high throughput sequencing (DamIP-seq). The DamIP-seq-defined hERα cistrome identifies many new binding regions and overlaps with those determined by chromatin immunoprecipitation (ChIP)-chip or ChIP-seq. Elements uniquely identified by DamIP-seq include a unique class of elements that show low, but persistent, hERα binding when reexamined by conventional ChIP. In contrast, DamIP-seq fails to detect some elements with very transient hERα binding. The methyl-adenine modifications introduced by Dam are stable and do not decrease over 12 d. In summary, the current study provides both an alternate view of the hERα cistrome to further understand the mechanism of hERα-mediated transcription and a new tool to explore other transcriptional factors and cofactors that is very different from conventional ChIP.
Assuntos
Proteínas de Escherichia coli/biossíntese , Receptor alfa de Estrogênio/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , DNA Metiltransferases Sítio Específica (Adenina-Específica)/biossíntese , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Proteínas de Escherichia coli/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Elementos Reguladores de Transcrição , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismoRESUMO
FABP4 delivers specific ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby facilitating the ligation and enhancing the transcriptional activity of the receptor. Here, we delineate the structural features that underlie the nucleocytoplasmic transport of FABP4. The primary sequence of FABP4 does not harbor a readily identifiable nuclear localization signal (NLS). However, such a signal could be found in the three-dimensional structure of the protein and was mapped to three basic residues that form a functional NLS stabilized by the FABP4/PPARgamma ligand troglitazone. We show that FABP4 is also subject to active nuclear export. Similarly to the NLS, the nuclear export signal (NES) is not apparent in the primary sequence, but assembles in the tertiary structure from three nonadjacent leucine residues to form a motif reminiscent of established NES. The data demonstrate that both nuclear export and nuclear import are critical for enabling FABP4 to enhance the transcriptional activity of PPARgamma. Additionally, the observations provide insight into the fundamental question of how proteins are activated by ligands. Such an activation may be understood by the "induced-fit" model, which states that ligand-induced conformational changes precede activation of a protein. Alternatively, the "pre-existing equilibrium" hypothesis postulates that activated conformations exist within the repertoire of apoproteins, and that ligands do not induce these but merely stabilize them. Studies of the subcellular localization of FABP4 support the validity of the "pre-existing equilibrium" model for the ligand-controlled activation of the nuclear import of FABP4.