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1.
Chemosphere ; 262: 128356, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182092

RESUMO

Polybrominated diphenyl ethers (PBDEs) are often suspected to activate the signal transduction pathway of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, for the induction of toxicity. Hence, the binding property of PBDEs with AhR is assumed to be associated with the ligand-dependent activation of AhR that may introduce many drug-metabolizing enzymes of genes encoding. However, the binding mechanism and the structural effect of PBDEs on their binding properties of AhR still need to be unraveled for toxicology research. A comprehensive study of the PBDEs-AhR binding mechanism was investigated using an integrated molecular modeling approach with two-dimensional quantitative structure-activity relationships (2D-QSAR), three-dimensional QSAR (3D-QSAR), and molecular docking simulation. Molecular docking revealed the differences in binding domains among 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-AhR complex and two PBDE-AhR complexes. A 2D-QSAR model was developed to analyze the overall structural effects of PBDEs on the binding affinity of AhR. It provided an insight into major physico-chemical properties by multiple linear regression based on genetic algorithm with reasonable results. The 3D-QSAR modeling discovered the detailed interaction features of binding sites, configurations and interaction fields of AhR with different PBDE ligands. This study demonstrated that the descriptors of Smin69 and MoRSEC15 were related to electronic properties and had a great effect on the relative binding affinities. The position of Br substitutions exhibited a significant influence on the interactions between AhR and PBDEs, including halogen interaction, π-S interaction, π-π stacking interaction, and hydrophobic effect. This integrated molecular modeling approach provided a comprehensive analysis of the structural effects of PBDEs on their binding properties with AhR at molecular level.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Éteres Difenil Halogenados/química , Éteres Difenil Halogenados/metabolismo , Dibenzodioxinas Policloradas , Relação Quantitativa Estrutura-Atividade , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/metabolismo , Transdução de Sinais
2.
Development ; 147(21)2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32907848

RESUMO

The closure of the embryonic ventral body wall in amniotes is an important morphogenetic event and is essential for life. Defects in human ventral wall closure are a major class of birth defect and a significant health burden. Despite this, very little is understood about how the ventral body wall is formed. Here, we show that fibroblast growth factor (FGF) ligands FGF8, FGF17 and FGF18 are essential for this process. Conditional mouse mutants for these genes display subtle migratory defects in the abdominal muscles of the ventral body wall and an enlarged umbilical ring, through which the internal organs are extruded. By refining where and when these genes are required using different Cre lines, we show that Fgf8 and Fgf17 are required in the presomitic mesoderm, whereas Fgf18 is required in the somites. This study identifies complex and multifactorial origins of ventral wall defects and has important implications for understanding their origins during embryonic development.


Assuntos
Padronização Corporal , Embrião de Mamíferos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/genética , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Hérnia Umbilical , Masculino , Camundongos , Modelos Biológicos , Morfogênese , Mutação/genética , Especificidade de Órgãos , Domínios Proteicos , Somitos/metabolismo , Células-Tronco/metabolismo
3.
Mutat Res ; 785: 108319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32800270

RESUMO

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fenda Labial/genética , Fissura Palatina/genética , Proteínas Repressoras/genética , Fumar/efeitos adversos , Motivos de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Metilação de DNA , Suplementos Nutricionais , Feminino , Ácido Fólico/metabolismo , Estudos de Associação Genética , Humanos , Recém-Nascido , Masculino , Modelos Moleculares , Domínios Proteicos , Isoformas de RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fatores de Risco
4.
PLoS Genet ; 16(5): e1008797, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392219

RESUMO

Sun-loving plants perceive the proximity of potential light-competing neighboring plants as a reduction in the red:far-red ratio (R:FR), which elicits a suite of responses called the "shade avoidance syndrome" (SAS). Changes in R:FR are primarily perceived by phytochrome B (phyB), whereas UV-B perceived by UV RESISTANCE LOCUS 8 (UVR8) elicits opposing responses to provide a counterbalance to SAS, including reduced shade-induced hypocotyl and petiole elongation. Here we show at the genome-wide level that UVR8 broadly suppresses shade-induced gene expression. A subset of this gene regulation is dependent on the UVR8-stabilized atypical bHLH transcription regulator LONG HYPOCOTYL IN FAR-RED 1 (HFR1), which functions in part redundantly with PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1). In parallel, UVR8 signaling decreases protein levels of the key positive regulators of SAS, namely the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5, in a COP1-dependent but HFR1-independent manner. We propose that UV-B antagonizes SAS via two mechanisms: degradation of PIF4 and PIF5, and HFR1- and PIL1-mediated inhibition of PIF4 and PIF5 function. This work highlights the importance of typical and atypical bHLH transcription regulators for the integration of light signals from different photoreceptors and provides further mechanistic insight into the crosstalk of UVR8 signaling and SAS.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Raios Ultravioleta/efeitos adversos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Estabilidade Proteica , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
Int J Mol Sci ; 21(7)2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32252465

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transformação Celular Neoplásica/metabolismo , DNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Transformação Celular Neoplásica/genética , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligantes , Modelos Moleculares , Molibdênio/farmacologia , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Hidrocarboneto Arílico/química , Relação Estrutura-Atividade
6.
Mol Immunol ; 120: 164-178, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32146146

RESUMO

The human hs1.2 enhancer within the Ig heavy chain gene (IGH) is polymorphic and associated with a number of autoimmune diseases. The polymorphic region is characterized by tandem repeats of an ∼53-bp invariant sequence containing possible binding sites for several transcription factors. Our previous studies suggest the human hs1.2 enhancer is sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and high affinity ligand of the aryl hydrocarbon receptor (AhR). TCDD induced hs1.2 enhancer activity in an AhR-dependent manner and the number of invariant sequences influenced the magnitude of activity. To better understand the regulation of human hs1.2 enhancer activity, the objective of the current study was to utilize mutational analysis and luciferase reporter constructs to evaluate the contribution of putative transcription factor binding sites to overall hs1.2 enhancer activity and modulation by TCDD. Basal and LPS-induced activity of the hs1.2 enhancer appeared to be most affected by mutation of sites outside of the invariant sequence or deletion of the entire invariant sequence; whereas sites influencing the effect of TCDD were dependent on the cellular activation state (i.e. unstimulated vs. LPS stimulation) and relatively independent of the putative AhR binding site within the invariant sequence. These results suggest that AhR activation affects human hs1.2 activity through an as yet undetermined non-canonical pathway. A better understanding regarding the role of the hs1.2 enhancer in human Ig expression and how AhR ligands modulate its activity may lead to insights into overall Ig regulation and mechanisms of dysfunction.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação/genética , Linhagem Celular , Elementos Facilitadores Genéticos/efeitos dos fármacos , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Ativação Transcricional/efeitos dos fármacos
7.
Proc Natl Acad Sci U S A ; 117(8): 4199-4210, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029586

RESUMO

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição SOXE/metabolismo , Raízes Nervosas Espinhais/metabolismo , Motivos de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Embrião de Galinha , Proteínas F-Box/química , Proteínas F-Box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Crista Neural/citologia , Crista Neural/metabolismo , Neurogênese , Ligação Proteica , Estabilidade Proteica , Raízes Nervosas Espinhais/citologia
8.
Biochemistry ; 59(4): 450-459, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31868344

RESUMO

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that are induced in many cancer types, promoting tumor-forming pathways. The HIF-2α PAS-B domain of the human HIF-2 complex contains a large buried hydrophilic cavity that is filled with eight ordered water molecules, making the domain a promising drug target. Recent work has led to the hypothesis that these buried water molecules act as an internal scaffold that stabilizes the buried cavity, holding it open to ease ligand binding. Here we show that extensive molecular dynamics simulations of the HIF-2α PAS-B domain strongly support this hypothesis. If water is prevented from entering the buried cavity, several amino acid side chains lining the cavity undergo a major conformational change that shrinks the cavity volume by an average of ∼45% relative to the hydrated cavity. The structural change within the cavity is due mainly to side chain rearrangements; the absence of bound water has only minor effects on the protein backbone conformation. Moreover, when the cavity lacks internally bound water, the side chain of Tyr307 rotates such that it blocks a major route for entry of the ligand into the cavity, greatly raising the free energy barrier for binding of an artificial ligand compared to when the cavity contains water. Finally, there is no difference in global protein stability of PAS-B when the cavity lacks water, as indicated by the same degree of protein flexibility and identical predicted melting temperatures. These findings help to explain further how water stabilizes large internal cavities, a relatively underexamined feature of proteins.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fenômenos Biofísicos , Cristalografia por Raios X , Dimerização , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Água/química
9.
New Phytol ; 225(1): 250-267, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31487399

RESUMO

The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bioensaio/métodos , Mutação/genética , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Células CHO , Cricetinae , Cricetulus , Modelos Biológicos , Fosforilação , Fosfotirosina/metabolismo , Multimerização Proteica , Estabilidade Proteica , Ativação Transcricional/genética
10.
Theor Appl Genet ; 133(1): 317-328, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31637460

RESUMO

KEY MESSAGE: A DNA transposon was found in the gene encoding a bHLH transcription factor. Genotypes of the marker tagging this DNA transposon perfectly co-segregated with color phenotypes in large F2:3 populations A combined approach of bulked segregant analysis and RNA-Seq was used to isolate causal gene for C locus controlling white bulb color in onions (Allium cepa L.). A total of 114 contigs containing homozygous single nucleotide polymorphisms (SNPs) between white and yellow bulked RNAs were identified. Four of them showed high homologies with loci clustered in the middle of chromosome 5. SNPs in 34 contigs were confirmed by sequencing of PCR products. One of these contigs showed perfect linkage to the C locus in F2:3 populations consisting of 2491 individuals. However, genotypes of molecular marker tagging this contig were inconsistent with color phenotypes of diverse breeding lines. A total of 146 contigs showed differential expression between yellow and white bulks. Among them, transcription levels of B2 gene encoding a bHLH transcription factor were significantly reduced in white RNA bulk and F2:3 individuals, although there was no SNP in the coding region. Phylogenetic analysis showed that onion B2 was orthologous to bHLH-coding genes regulating anthocyanin biosynthesis pathway in other plant species. Promoter regions of B2 gene were obtained by genome walking and a 577-bp non-autonomous DNA transposon designated as AcWHITE was found in the white allele. Molecular marker tagging AcWHITE showed perfect linkage with the C locus. Marker genotypes of the white allele were detected in some white accessions. However, none of tested red or yellow onions contained AcWHITE insertion, implying that B2 gene was likely to be a casual gene for the C locus.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Elementos de DNA Transponíveis/genética , Genes de Plantas , Cebolas/genética , Pigmentação/genética , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Cromossomos de Plantas/genética , DNA de Plantas/genética , Dosagem de Genes , Ligação Genética , Loci Gênicos , Marcadores Genéticos , Genótipo , Padrões de Herança/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , RNA de Plantas/genética , Transcrição Genética
11.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671600

RESUMO

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Orchidaceae/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Clonagem Molecular , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Orchidaceae/genética , Orchidaceae/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Multimerização Proteica , Ativação Transcricional
12.
Genes (Basel) ; 10(10)2019 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614829

RESUMO

Development requires the careful orchestration of several biological events in order to create any structure and, eventually, to build an entire organism. On the other hand, the fate transformation of terminally differentiated cells is a consequence of erroneous development, and ultimately leads to cancer. In this review, we elaborate how development and cancer share several biological processes, including molecular controls. Transcription factors (TF) are at the helm of both these processes, among many others, and are evolutionarily conserved, ranging from yeast to humans. Here, we discuss four families of TFs that play a pivotal role and have been studied extensively in both embryonic development and cancer-high mobility group box (HMG), GATA, paired box (PAX) and basic helix-loop-helix (bHLH) in the context of their role in development, cancer, and their conservation across several species. Finally, we review TFs as possible therapeutic targets for cancer and reflect on the importance of natural resistance against cancer in certain organisms, yielding knowledge regarding TF function and cancer biology.


Assuntos
Desenvolvimento Embrionário , Neoplasias/metabolismo , Fatores de Transcrição/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 , Movimento Celular/genética , Movimento Celular/imunologia , Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição GATA/química , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Proteínas HMGB/química , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética
13.
Int J Mol Sci ; 20(21)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31653121

RESUMO

The bHLH proteins are a family of eukaryotic transcription factors regulating expression of a wide range of genes involved in cell differentiation and development. They contain the Helix-Loop-Helix (HLH) domain, preceded by a stretch of basic residues, which are responsible for dimerization and binding to E-box sequences. In addition to the well-preserved DNA-binding bHLH domain, these proteins may contain various additional domains determining the specificity of performed transcriptional regulation. According to this, the family has been divided into distinct classes. Our aim was to emphasize the significance of existing disordered regions within the bHLH transcription factors for their functionality. Flexible, intrinsically disordered regions containing various motives and specific sequences allow for multiple interactions with transcription co-regulators. Also, based on in silico analysis and previous studies, we hypothesize that the bHLH proteins have a general ability to undergo spontaneous phase separation, forming or participating into liquid condensates which constitute functional centers involved in transcription regulation. We shortly introduce recent findings on the crucial role of the thermodynamically liquid-liquid driven phase separation in transcription regulation by disordered regions of regulatory proteins. We believe that further experimental studies should be performed in this field for better understanding of the mechanism of gene expression regulation (among others regarding oncogenes) by important and linked to many diseases the bHLH transcription factors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dimerização , Regulação da Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Transcrição Genética
14.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554340

RESUMO

The bHLH-PAS (basic helix-loop-helix/ Period-ARNT-Single minded) proteins are a family of transcriptional regulators commonly occurring in living organisms. bHLH-PAS members act as intracellular and extracellular "signals" sensors, initiating response to endo- and exogenous signals, including toxins, redox potential, and light. The activity of these proteins as transcription factors depends on nucleocytoplasmic shuttling: the signal received in the cytoplasm has to be transduced, via translocation, to the nucleus. It leads to the activation of transcription of particular genes and determines the cell response to different stimuli. In this review, we aim to present the current state of knowledge concerning signals that affect shuttling of bHLH-PAS transcription factors. We summarize experimentally verified and published nuclear localization signals/nuclear export signals (NLSs/NESs) in the context of performed in silico predictions. We have used most of the available NLS/NES predictors. Importantly, all our results confirm the existence of a complex system responsible for protein localization regulation that involves many localization signals, which activity has to be precisely controlled. We conclude that the current stage of knowledge in this area is still not complete and for most of bHLH-PAS proteins an experimental verification of the activity of further NLS/NES is needed.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Família Multigênica , Sinais de Localização Nuclear , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-Atividade
15.
Proc Natl Acad Sci U S A ; 116(40): 19911-19916, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527239

RESUMO

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.


Assuntos
Proteínas CLOCK/química , Elementos E-Box , Heme/química , Transdução de Sinais , Fatores de Transcrição ARNTL/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Catálise , Relógios Circadianos , Criptocromos/química , DNA/química , Elétrons , Escherichia coli/metabolismo , Humanos , Ligantes , Proteínas do Tecido Nervoso/química , Oxigênio/química , Proteínas Circadianas Period/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Transcrição Genética
16.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
17.
Nat Commun ; 10(1): 3293, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337753

RESUMO

Duplication of ancestral hypoxia-inducible factor (HIF)α coincided with the evolution of vertebrate species. Paralogs HIF1α and HIF2α are the most well-known factors for modulating the cellular transcriptional profile following hypoxia. However, how the processes of natural selection acted upon the coding region of these two genes to optimize the cellular response to hypoxia during evolution remains unclear. A key negative regulator of HIFα is von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that evolutionarily-relevant substitutions can modulate a secondary contact between HIF1α Met561 and VHL Phe91. Notably, HIF1α binds more tightly than HIF2α to VHL due to a conserved Met to Thr substitution observed in the vertebrate lineage. Similarly, substitution of VHL Phe91 with Tyr, as seen in invertebrate species, decreases VHL affinity for both HIF1α and HIF2α. We propose that vertebrate evolution involved a more complex hypoxia response with fine-tuned divergence of VHL affinity for HIF1α and HIF2α.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Evolução Biológica , Duplicação Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Substituição de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Evolução Molecular , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metionina/metabolismo , Oxirredução , Oxigênio/metabolismo , Seleção Genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
18.
Int J Mol Sci ; 20(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357385

RESUMO

The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a class of transcriptional regulators, commonly occurring in living organisms and highly conserved among vertebrates and invertebrates. These proteins exhibit a relatively well-conserved domain structure: the bHLH domain located at the N-terminus, followed by PAS-A and PAS-B domains. In contrast, their C-terminal fragments present significant variability in their primary structure and are unique for individual proteins. C-termini were shown to be responsible for the specific modulation of protein action. In this review, we present the current state of knowledge, based on NMR and X-ray analysis, concerning the structural properties of bHLH-PAS proteins. It is worth noting that all determined structures comprise only selected domains (bHLH and/or PAS). At the same time, substantial parts of proteins, comprising their long C-termini, have not been structurally characterized to date. Interestingly, these regions appear to be intrinsically disordered (IDRs) and are still a challenge to research. We aim to emphasize the significance of IDRs for the flexibility and function of bHLH-PAS proteins. Finally, we propose modern NMR methods for the structural characterization of the IDRs of bHLH-PAS proteins.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Biologia Computacional/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Anatômicos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Relação Estrutura-Atividade
19.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140252, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31325636

RESUMO

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 µM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R1, R1ρ, R2 and ηxy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , DNA/química , Complexos Multiproteicos/química , Proteínas de Neoplasias/química , Precursores de Proteínas/química , Timosina/análogos & derivados , Humanos , Domínios Proteicos , Espalhamento a Baixo Ângulo , Timosina/química , Difração de Raios X
20.
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
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