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1.
Nat Commun ; 10(1): 2835, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249377

RESUMO

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Proteínas Repressoras/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos/embriologia , Camundongos/metabolismo , Camundongos Endogâmicos ICR , Camundongos Knockout , Células-Tronco Neurais/química , Células-Tronco Neurais/citologia , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Célula Única
2.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
3.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934833

RESUMO

Mineralocorticoid receptor (MR)-mediated signaling in the brain has been suggested as a protective factor in the development of psychopathology, in particular mood disorders. We recently identified genomic loci at which either MR or the closely related glucocorticoid receptor (GR) binds selectively, and found members of the NeuroD transcription factor family to be specifically associated with MR-bound DNA in the rat hippocampus. We show here using forebrain-specific MR knockout mice that GR binding to MR/GR joint target loci is not affected in any major way in the absence of MR. Neurod2 binding was also independent of MR binding. Moreover, functional comparison with MyoD family members indicates that it is the chromatin remodeling aspect of NeuroD, rather than its direct stimulation of transcription, that is responsible for potentiation of MR-mediated transcription. These findings suggest that NeuroD acts in a permissive way to enhance MR-mediated transcription, and they argue against competition for DNA binding as a mechanism of MR- over GR-specific binding.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Corticosterona/metabolismo , DNA/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Knockout , Modelos Biológicos , Proteína MyoD/metabolismo , Proteínas do Tecido Nervoso/química , Ligação Proteica , Domínios Proteicos , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade
4.
Gene ; 702: 89-98, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30928363

RESUMO

Teosinte branched 1/Cycloidea/Proliferating cell factor 1 (TCP) proteins belongs to a plant-specific transcription factor family that plays important roles in plant development. TCP gene-regulated plant branching occurs downstream in the strigolactone pathway. In this study, 41 TCP genes were identified in the genome of Panicum virgatum L. (switchgrass). These genes all contained the TCP conserved domain, and they belonged to two subfamilies distributed across 18 chromosomes. Analysis of gene expression using RNA-Seq data showed that 16 TCP genes were highly expressed in the inflorescence and shoot. The expression patterns of 13 selected PvTCP genes were analyzed in different tissues, and their responses to strigolactones (SLs) were examined. The selected genes were expressed differentially in a range of tissues and to application of SLs, indicating that PvTCPs were involved in a range of developmental and physiological processes. This genome-wide analysis and determination of PvTCP gene-expression patterns yielded valuable information on switchgrass development that will inform studies into improving switchgrass and other species for crop production.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Família Multigênica , Panicum/genética , Proteínas de Plantas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/classificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromossomos de Plantas , Genes de Plantas , Panicum/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Sintenia
5.
Nat Commun ; 10(1): 1459, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926794

RESUMO

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.


Assuntos
Linfoma de Burkitt/genética , Genoma Humano , Transcriptoma/genética , Adolescente , Processamento Alternativo/genética , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Estudos de Coortes , Metilação de DNA/genética , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação INDEL/genética , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequenciamento Completo do Genoma
6.
Gene ; 690: 113-119, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30593917

RESUMO

MicroRNA is an important regulation factor in insect development and metamorphosis. It has been reported that E(spl)m4 is a miRNA-targeted gene, as well as the target of the Notch signaling pathway in Drosophila. The expression of E(spl)m4 can be regulated by microRNA and further affect the neural development of Drosophila. Here, we found that BmEm4, an ortholog of E(spl)m4 from Bombyx mori, was the target gene of bmo-miR-79, with target sites containing the Brd and K boxes of the BmEm4_3'UTR, which was validated by the dual luciferase reporter (DLR) assay. Furthermore, bmo-miR-79 mimics can inhibit the expression of BmEm4 in BmN cells after transfection, and bmo-miR-79 can also inhibit the expression of BmEm4 in different developmental stages of Bombyx mori at a posttranscriptional level, to different degrees. The EMSA test further showed that bmo-miR-79 could bind to BmAGO2, which is the Bombyx mori argonaute2 protein, suggesting that bmo-miR-79 might regulate the expression of BmEm4 by forming miRISC complexes with BmAGO2. Taken together, bmo-miR-79 could regulate the expression of BmEm4 mediated by BmAGO2 and further affect its function in the silkworm Bombyx mori.


Assuntos
Proteínas Argonauta/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bombyx/crescimento & desenvolvimento , Regulação para Baixo , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Bombyx/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Metamorfose Biológica , Transdução de Sinais
7.
Drug Discov Today ; 23(7): 1444-1451, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29753878

RESUMO

Hypoxia-inducible factors (HIF-1α, -2α -3α, and -ß) are key factors that control hypoxia-induced carcinogenic pathways. HIF-1α is predominantly involved in the early stages of cancer, whereas HIF-2α is actively involved in the later stages; in addition, chronic (prolonged) rather than acute (short) hypoxia is a feature of metastasis and chemoresistance that occur during the later stages of cancer. Oncometabolites, onco-miRNAs, glucose deprivation, pseudohypoxia, cytokine/chemokine secretion, and some unique upstream proteins are involved in the signaling switch from HIF-1α to HIF-2α; thus, understanding this signaling switch is critical for the treatment of advanced cancer. In this review, we highlight data relating to HIF-2α rather than HIF-1α signaling in cancer pathways and discuss prospective drugs that target this important factor.


Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Desenho de Drogas , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Hipóxia Tumoral , Microambiente Tumoral
8.
Biochem Biophys Res Commun ; 500(2): 443-449, 2018 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-29660343

RESUMO

Although hypoxia-inducible factor (HIF) 1α and 2α function as master regulators of the transcriptional response to hypoxia, the function of HIF3α and its responses to hypoxic stress remain unclear in teleost fish. Here, we characterized the HIF3α cDNA in hypoxia-sensitive blunt snout bream (Megalobrama amblycephala), with 3059 bp length, consisting of an open reading frame (ORF) encoding 643 amino acid residues. Blunt snout bream HIF3α mRNA was stably expressed during stages of embryonic development and in adult tissues. After a 4 h hypoxia stress, HIF3α mRNA of the juvenile fish was significantly upregulated in the liver, brain, and kidney, and restored to the pretreatment levels after a 24 h recovery. When tagged with enhanced green fluorescent protein (EGFP) and transfected into cultured HeLa cells, blunt snout bream HIF3α was mainly distributed in the nucleus under normoxia. Treatment of the cells with CoCl2 to mimic hypoxic conditions showed that there was no effect about the nuclear localization of HIF3α but a statistically significant increase in HIF3α protein levels. A nuclear localization signal (NLS) sequence at the C-terminus of HIF3α may exert positive effects in the process of nuclear localization. These results suggest that blunt snout bream HIF3α could be involved in different physiological functions under normoxia and hypoxia conditions.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Cyprinidae/genética , Hipóxia/metabolismo , Transcrição Genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Células HeLa , Humanos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
9.
PLoS Comput Biol ; 14(2): e1006021, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29489822

RESUMO

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Animais , Cristalografia por Raios X , Ligantes , Camundongos , Mutação , Oscilometria , Oxigênio/química , Ligação Proteica , Multimerização Proteica , Termodinâmica , Transcrição Genética , Água/química
10.
Biochim Biophys Acta Gen Subj ; 1862(6): 1283-1295, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29530795

RESUMO

BACKGROUND: NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions. METHODS: We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction. RESULTS: Peptide dissociation constants towards wild-type NUPR1 were ~ 3 µM, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B). CONCLUSIONS: Designed peptides can be used as lead compounds to inhibit NUPR1 interactions. GENERAL SIGNIFICANCE: Peptides may be exploited as drugs to target IDPs.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Capsídeo/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Proteínas de Neoplasias/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
11.
Nat Commun ; 9(1): 575, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422508

RESUMO

One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doxorrubicina/administração & dosagem , Células Endoteliais/metabolismo , Neoplasias/tratamento farmacológico , Superóxido Dismutase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/genética , Caderinas/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Tratamento Farmacológico , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica , Superóxido Dismutase/genética
12.
Proc Natl Acad Sci U S A ; 115(10): E2238-E2245, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29463750

RESUMO

Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criptocromos/metabolismo , Citoplasma/efeitos da radiação , Fotorreceptores Microbianos/química , Ligação Proteica/efeitos da radiação , Animais , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criptocromos/química , Criptocromos/genética , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Células HeLa , Humanos , Cinética , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Neurospora crassa/química , Neurospora crassa/metabolismo , Neurospora crassa/efeitos da radiação , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Multimerização Proteica/efeitos da radiação
13.
J Mol Recognit ; 31(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28990233

RESUMO

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors related with the onset and progression of solid tumors. Studies demonstrated a class of tetrazole containing chiral inhibitors could stereoselectively disrupt the HIF-2 dimerization and reduce the target gene expression. However, the dynamical features and structural motifs of the HIF-2 heterodimer caused by the binding of enantiomers have not been rationalized at the atomistic level. In this work, molecular dynamics (MD) simulations combined with adaptive steered MD (ASMD) simulations were used to investigate stereoselective interrupting mechanism of HIF-2. Our results decipher that the binding of ligand A (S, R)-24 begets the significant conformation changes of ß-sheets and interrupts the HIF-2α/ARNT heterodimerization, which may be attributed to the disruption of the hydrogen bond and salt bridge interactions formed by the 4 foremost residues (Asp240, Arg247, Glu362, and Arg366) and the destruction of hydrophobic interactions on the binding interface. By contrast, the binding of ligand B (R, S)-24 does not disrupt protein dimerization and causes the motion of Fα helix in HIF-2α PAS-B domain to further change the major tunnel for ligand ingress and engress. The present work provides important molecular-level insight into the effect of the binding enantiomers on HIF-2 heterodimerization and bridges the gap between theory and the experimental results, which may conduce to develop highly potent antagonists for intervening the HIF-2-driven tumors.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Motivos de Aminoácidos , Dimerização , Humanos , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estereoisomerismo , Termodinâmica
14.
Pediatr Diabetes ; 19(2): 236-242, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28664602

RESUMO

AIMS: Only a few families with neuronal differentiation 1 (NEUROD1)-deficient diabetes, currently designated as maturity-onset diabetes of the young 6 (MODY6), have been reported, but mostly in Caucasian, and no mutation has been identified by family-based screening in Japanese. Accordingly, the phenotypic details of the disease remain to be elucidated. METHODS: We examined a total of 275 subjects having diabetes suspected to be MODY who were negative for mutations in MODY1-5 referred from 155 medical institutions throughout Japan. So as not to miss low penetrant cases, we examined non-obese Japanese patients with early-onset diabetes regardless of the presence of family history by direct sequencing of all exons and flanking regions of NEUROD1 . Large genomic rearrangements also were examined. RESULTS: Four patients with 3 frameshift mutations and 1 missense mutation, all of which were heterozygous and 3 of which were novel, were identified. Diabetic ketosis was found occasionally in these patients even under conditions of chronic hyperglycemia, for unknown reasons. Although the capacity of early-phase insulin secretion was low in these patients, the insulin secretory capacity was relatively preserved compared to that in hepatocyte nuclear factor (HNF)1A- and HNF1B-MODY. One of the patients and 2 of their diabetic mothers were found to have some mental or neuronal abnormality. CONCLUSIONS: This is the first report of NEUROD1 mutations in Japanese, who have a genetic background of intrinsically lower capacity of insulin secretion. NEUROD1-deficient diabetes appears to be low penetrant, and may occur in concert with other genetic factors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diabetes Mellitus Tipo 2/genética , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Cetoacidose Diabética/etiologia , Éxons , Feminino , Mutação da Fase de Leitura , Frequência do Gene , Heterozigoto , Humanos , Hiperglicemia/etiologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Japão , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/etiologia , Linhagem , Penetrância , Adulto Jovem
15.
J Environ Pathol Toxicol Oncol ; 37(4): 331-339, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30806239

RESUMO

The hypoxic tumor microenvironment is one of the major causes of the enhanced chemoresistant and radioresistant behavior of cancer cells. Therefore, the hypoxia-induced factor (HIF) pathway can be endorsed, for not only the malignant phenotype of the cells, but also its metastatic potential. Many drugs targeting the HIF pathways have failed in the clinical setting to demonstrate therapeutic efficacy. Such failures occur due to lack of specificity or redundancy in the complexity of tumor signaling/metabolism that can overcome the inhibitory effects. Another important factor is the letdown of the compound that can be accredited to lack of patient selection in the trials. Although many clinical trials have evaluated the efficacy of anticancer therapeutics and examined their effects on HIF levels, patients were not selected based on their HIF expression levels. If patients do not have elevated levels of HIF, then the therapeutics that target the HIF pathway may be less effective. In the present work, we have targeted HIF-2α of the HIF pathway. Ellagic acid (EA), a well-known anticancer compound and radiosensitizer, is used to inhibit the activity of HIF-2α. Our results show a very unique binding of EA with HIF-2α. Such new agents should be used in combination therapy and will hopefully overcome the resistance that may develop during initial treatment if the patient is identified to have enhanced expression of HIF-2α. Molecular dynamics studies followed solvation free energy calculations (molecular mechanics Poisson-Boltzmann surface area) for understanding the binding stability and per residue contribution. Our in silico data look promising and EA should be studied more in in vitro and in vivo for further analysis of its efficacy.


Assuntos
Antineoplásicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Ácido Elágico/metabolismo , Modelos Moleculares , Antineoplásicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Simulação por Computador , Ácido Elágico/química , Humanos
16.
PLoS One ; 12(12): e0190241, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29281714

RESUMO

Survival of cancer cells in the harsh tumor microenvironment, characterized by oxygen and glucose deprivation, requires rapid initiation of cytoprotective measures. Metabolites whose levels change during stress are ideal signaling cues, particularly if used in post-translational modifications of stress-responsive signal transducers. In cancer cells exposed to oxygen or glucose deprivation, there is an increase in cellular levels of acetate, a substrate for acetate-dependent acetyl CoA synthetase 2 (Acss2) that also stimulates translocation of Acss2 from the cytosol to the nucleus. Nuclear, but not cytosolic, Acss2 promotes acetylation of the stress-responsive Hypoxia Inducible Factor 2α (HIF-2α) subunit by the acetyltransferase/coactivator Creb binding protein (Cbp), a process that facilitates stable Cbp/HIF-2α complex formation. In addition to promoting de novo transcription, Cbp and HIF-2α act in concert to regulate local histone 3 epigenetic marks. Exogenous acetate augments Acss2/HIF-2 dependent cancer growth and metastasis in cell culture and mouse models. Thus, an acetate switch in mammals links nutrient intake and stress signaling with tumor growth and metastasis.


Assuntos
Acetato-CoA Ligase/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epigênese Genética , Neoplasias/metabolismo , Estresse Oxidativo , Transdução de Sinais , Acetilcoenzima A/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Linhagem Celular Tumoral , Células HEK293 , Humanos , Lisina/metabolismo , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Reação em Cadeia da Polimerase em Tempo Real
17.
Mol Microbiol ; 106(6): 876-890, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28994223

RESUMO

Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Genes Fúngicos/fisiologia , Fosfolipídeos/biossíntese , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Colina/metabolismo , DNA Fúngico , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Inositol/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Fúngico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transcrição Genética
18.
J Biol Chem ; 292(43): 17609-17616, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28904176

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Multimerização Proteica , Proteínas Repressoras/química , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Transcrição Genética/fisiologia
19.
J Pharmacol Exp Ther ; 363(3): 336-347, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28928122

RESUMO

Decreased erythropoietin (EPO) production, shortened erythrocyte survival, and other factors reducing the response to EPO contribute to anemia in patients who have a variety of underlying pathologies such as chronic kidney disease. Treatment with recombinant human EPO (rHuEPO) at supraphysiologic concentrations has proven to be efficacious. However, it does not ameliorate the condition in all patients, and it presents its own risks, including cardiovascular complications. The transcription factors hypoxia-inducible factor (HIF) 1α and HIF2α control the physiologic response to hypoxia and invoke a program of increased erythropoiesis. Levels of HIFα are modulated by oxygen tension via the action of a family of HIF-prolyl hydroxylases (PHDs), which tag HIFα for proteasomal degradation. Inhibition of these PHDs simulates conditions of mild hypoxia, leading to a potentially more physiologic erythropoietic response and presenting a potential alternative to high doses of rHuEPO. Here we describe the discovery and characterization of GSK1278863 [2-(1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido) acetic acid], a pyrimidinetrione-glycinamide low nanomolar inhibitor of PHDs 1-3 that stabilizes HIFα in cell lines, resulting in the production of increased levels of EPO. In normal mice, a single dose of GSK1278863 induced significant increases in circulating plasma EPO but only minimal increases in plasma vascular endothelial growth factor (VEGF-A) concentrations. GSK1278863 significantly increased reticulocytes and red cell mass parameters in preclinical species after once-daily oral administration and has demonstrated an acceptable nonclinical toxicity profile, supporting continued clinical development. GSK1278863 is currently in phase 3 clinical trials for treatment of anemia in patients with chronic kidney disease.


Assuntos
Barbitúricos/farmacologia , Drogas em Investigação/farmacologia , Inibidores Enzimáticos/farmacologia , Eritropoese/efeitos dos fármacos , Eritropoetina/agonistas , Glicina/análogos & derivados , Hematínicos/farmacologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Animais , Barbitúricos/administração & dosagem , Barbitúricos/efeitos adversos , Barbitúricos/farmacocinética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Cães , Relação Dose-Resposta a Droga , Drogas em Investigação/administração & dosagem , Drogas em Investigação/efeitos adversos , Drogas em Investigação/farmacocinética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Glicina/administração & dosagem , Glicina/efeitos adversos , Glicina/farmacocinética , Glicina/farmacologia , Hematínicos/administração & dosagem , Hematínicos/efeitos adversos , Hematínicos/farmacocinética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/agonistas , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Testes de Toxicidade Crônica
20.
BMC Genomics ; 18(1): 619, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28810832

RESUMO

BACKGROUND: As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae. RESULTS: A total of 146 bHLH TFs were identified in the Brachypodium distachyon genome and classified into 24 subfamilies. BdbHLHs in the same subfamily share similar protein motifs and gene structures. Gene duplication events showed a close relationship to rice, maize and sorghum, and segment duplications might play a key role in the expansion of this gene family. The amino acid sequence of the bHLH domains were quite conservative, especially Leu-27 and Leu-54. Based on the predicted binding activities, the BdbHLHs were divided into DNA binding and non-DNA binding types. According to the gene ontology (GO) analysis, BdbHLHs were speculated to function in homodimer or heterodimer manner. By integrating the available high throughput data in public database and results of quantitative RT-PCR, we found the expression profiles of BdbHLHs were different, implying their differentiated functions. CONCLUSION: One hundred fourty-six BdbHLHs were identified and their conserved domains, sequence features, phylogenetic relationship, chromosomal distribution, GO annotations, gene structures, gene duplication and expression profiles were investigated. Our findings lay a foundation for further evolutionary and functional elucidation of BdbHLH genes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Brachypodium/genética , Genômica , Proteínas de Plantas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Brachypodium/metabolismo , Cromossomos de Plantas/genética , Sequência Conservada , DNA de Plantas/metabolismo , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Multimerização Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Especificidade da Espécie
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