RESUMO
The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Predisposição Genética para Doença/genética , Proteínas Intrinsicamente Desordenadas/genética , Receptores de Hidrocarboneto Arílico/genética , Proteínas Repressoras/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutação de Sentido Incorreto , Processamento Pós-Transcricional do RNA , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Área Sob a Curva , Células COS , Chlorocebus aethiops , Simulação por Computador , Fluorescência , Hidrodinâmica , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic ß-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Células COS , Linhagem Celular , Chlorocebus aethiops , Sequências Hélice-Alça-Hélice , Camundongos , Modelos Moleculares , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Conformação Proteica , Domínios Proteicos , Transporte Proteico , RatosRESUMO
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-AtividadeRESUMO
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-AtividadeRESUMO
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 GênicaRESUMO
In this study Polish population data as well as efficiency parameters of 12 STR loci included in the Investigator HDplex set were presented. This set contains 9 systems not available in any other commercial multiplexes, ie.: D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325 and D21S2055. The evaluation was preformed based on DNA samples derived from 303 unrelated individuals living in Lodz region, central part of Poland. The obtained distribution of the genotypes is consistent with the assumptions of the Hardy and Weinberg equilibrium (HWE). It reflects properly genetic structure of the studied population compared with other populations of Europe and the world. It indicates the linkage equilibrium within the pairs of investigated loci, as well as with regard to other syntenic loci. The total value of the power of exclusion (PE) and the random match probability (MP) were respectively 0.99999988 and 5.2 × 10-18. Therefore the polymorphism of examined genetic markers within the Investigator HD-plex multiplex allows for a significant increase of the evidence value. Thus it constitutes an excellent tool for resolving difficult cases in the field of forensic genetics.
Assuntos
Impressões Digitais de DNA/métodos , Genética Populacional , Sequências de Repetição em Tandem/genética , Feminino , Frequência do Gene , Humanos , Masculino , Polônia , Reação em Cadeia da Polimerase , População Branca/genéticaRESUMO
A population data obtained on the basis of sample of 1000 unrelated individuals of Polish ancestry living in Lodz region of Central Poland with use of fluorescent multiplex-PCR and capillary electrophoresis were presented. Evaluation included 15 polymorphic loci DNA - STR from NGM multiplex-PCR set, ie. D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA. The allele frequency distribution and crucial statistical parameters for the investigated markers and the whole set were calculated. The compliance of the studied population with Hardy-Weinberg equilibrium, independence of inheritance and high parameters of the usefulness in forensic genetics have been demonstrated. The interpopulation comparison performed by the "neighbor-joining" method as well as multidimensional scaling depicted the genetic distances dividing the examined Polish population from other populations of Poland, Europe and the world.
Assuntos
Frequência do Gene , Loci Gênicos , Variação Genética , Sequências de Repetição em Tandem/genética , População Branca/genética , Alelos , Genética Populacional , Humanos , PolôniaRESUMO
The aim of this study was to assess the influence of bacterial biofilms in chronic rhinosinusitis (CRS) patients on the clinical outcomes following endoscopic sinus surgery (ESS). This was a prospective, double-blind study. Patients undergoing ESS, because of CRS, were recruited. Overall 80 patients were qualified. For each of these demographic, clinical and radiologic characteristics were recorded. During surgery each patient had at least 7 mucosal specimens taken to assess, using scanning electron microscopy, the possible presence of bacterial biofilms. Prior to mucosa specimen excision, swabs for bacteriological and fungal analyses were taken. Each patient underwent perioperative and follow-up assessment at 3 and 6 months post-ESS. Biofilms were found in 33 (41.3%) patients (study group). From among the 47 patients without the presence of biofilms, 33 (control group) were taken to match the study group in age, gender and clinical characteristics. The intensity of subjective and objective CRS symptoms, as well as patient quality-of-life, did not correlate with the Lund and Mackay score (p > 0.05). Analysis of variance showed that, in the control group or the group as a whole, the intensity of subjective and objective symptoms decreased (p < 0.05), and the quality-of-life increased with time (p < 0.05). In conclusion, biofilm-positive patients tend to have a greater severity of disease preoperatively and continue to have persistent and more severe symptoms post-ESS. This study supports the role of biofilms in maintaining the chronic and recalcitrant nature of CRS. The lack of planktonic bacteria in post-operative sinus swabs does not rule out the presence of bacterial biofilms.
Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Endoscopia , Fungos/fisiologia , Microscopia Eletrônica de Varredura , Complicações Pós-Operatórias/patologia , Rinite/patologia , Rinite/cirurgia , Sinusite/patologia , Sinusite/cirurgia , Adulto , Doença Crônica , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/microbiologia , Mucosa Nasal/patologia , Mucosa Nasal/cirurgia , Complicações Pós-Operatórias/microbiologia , Estudos Prospectivos , Qualidade de Vida , Rinite/microbiologia , Sinusite/microbiologia , Resultado do TratamentoRESUMO
Fuchs endothelial corneal dystrophy (FECD) is a slowly progressive eye disease leading to blindness, mostly affecting people above 40 years old. The only known method of curing FECD is corneal transplantation. The disease is characterized by the presence of extracellular deposits called "cornea guttata", apoptosis of corneal endothelial cells, dysfunction of Descement's membrane and corneal edema. Oxidative stress is suggested to play a role in FECD pathogenesis. Reactive oxygen species produced during the stress may damage biomolecules, including DNA. In the present study we evaluated the extent of endogenous DNA damage, including oxidatively modified DNA bases, and damage induced by hydrogen peroxide as well as the kinetics of DNA repair in peripheral blood mononuclear cells of 50 patients with FECD and 43 age-matched controls without visual disturbances. To quantify DNA damage and repair we used the alkaline comet assay technique with the enzymes recognizing oxidative DNA damage, hOGG1 and EndoIII. We did not observe differences in the extent of endogenous and hydrogen peroxide-induced DNA damage between FECD patients and controls. However, we found a lower efficacy of DNA repair in FECD patients as compared with control individuals. The results obtained suggest that the lowering of the DNA repair capacity may be one of the mechanisms underlying the role of oxidative stress in the FECD pathology.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Oftalmopatias/metabolismo , Distrofia Endotelial de Fuchs/genética , Apoptose/efeitos dos fármacos , Córnea/citologia , Córnea/metabolismo , Córnea/patologia , Edema da Córnea/genética , Edema da Córnea/metabolismo , Edema da Córnea/patologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Lâmina Limitante Posterior/metabolismo , Oftalmopatias/genética , Oftalmopatias/patologia , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patologia , Humanos , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
It is estimated that in Europe 10% of adults suffer from chronic sinusitis. Chronic sinusitis can be caused by many different diseases that share chronic inflammation of the sinuses as a common symptom. Rhinitis can be caused by stomach acid coming up from the stomach into the esophagus, which successively can result in chronic sinusitis. The current gold standard for diagnosing GERD is--bothersome for the patient--24 h esophageal pH monitoring. This method can be unpleasant for the patients, which makes it less acceptable. Because of that the criteria for symptomatic GERD were made an alternative diagnostic way. We acknowledge that the presence of heartburn and stomach acid coming up from the stomach into the esophagus at least once a week can be diagnosed as symptomatic GERD. The aim of the study is the assessment of the frequency of symptomatic GERD in patients operated because of chronic sinusitis and impact of symptomatic GERD on the follow-up treatment up to 12 months after endoscopic nasal surgery. The authors analysed 144 patients operated at the JUCM Otolaryngological Clinic in Kraków between 2011 and 2013 because of sinusitis. The inclusion criteria were: diagnosed chronic sinusitis, indications for endoscopic sinus surgery, and a written consent for the research. Each patient was examined laryngologically and surveyed. Patients were divided into two groups: with and without symptomatic GERD. We analysed the symptoms in patients treated for sinusitis with or without GERD before, between 3 and 6 as well as in the 12th month after endonasal surgery. Moreover, we analysed the intensity of the global symptoms (expressed in the VAS scale) and separately for each of the 13 symptoms of chronic sinusitis (expressed on a scale 0 - 3). We established that 33 out of the 144 patients (22.9%) qualified for the first survey reported the symptoms of GERD. In the second survey, which was conducted between 3 and 6 month after ESS, 24 out of 119 (20%) people reported the symptoms and in the third survey, which took place in the 12th month after ESS, 14 out of 52 patients reported symptomatic GERD. The intensity of global symptoms rated in the VAS scale in patients with chronic sinusitis during the first survey was 7.8 and in the second and third survey the intensity was 4.2 and 4.3 respectively. But in patients without any symptoms they were 7.4, 2.8, 3.2. We also analysed 13 symptoms of chronic sinusitis rated on a scale 0 - 3. The result of the research was that in patients with symptomatic GERD, even after FESS and the appropriate follow-up, we can still suspect such symptoms as streaming the fluid over the back side of the throat, cough, pain or the feeling of fullness in the ear, headache or halitosis. We should take it under consideration during qualification for the surgery as well as predicting the results of the treatment. Further research is required to state if and how different methods and procedures used in case of patients with symptomatic GERD can reduce the uncomfortable influence of this disease on the effects of chronic sinusitis treatment.
Assuntos
Refluxo Gastroesofágico/epidemiologia , Refluxo Gastroesofágico/cirurgia , Sinusite/epidemiologia , Adolescente , Adulto , Idoso , Criança , Doença Crônica , Comorbidade , Monitoramento do pH Esofágico , Esofagoscopia , Feminino , Seguimentos , Refluxo Gastroesofágico/diagnóstico , Refluxo Gastroesofágico/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância da População , Cuidados Pós-Operatórios/métodos , Sinusite/diagnóstico , Sinusite/cirurgia , Resultado do Tratamento , Adulto JovemRESUMO
For a long time, entomopathogenic fungi were considered alternative biological control factors. Recently, these organisms were shown to fulfill additional roles supporting plants' development, improving their resistance to disease and survival under stress conditions. Considering the documented interactions of B. bassiana with a wide range of plants, we aimed to evaluate the impact of aqueous extracts of the fungus on the growth of an agriculturally significant plant-wheat. The usage of fungal extracts instead of fungi could be beneficial especially in unfavorable, environmentally speaking, regions. Selected dilutions of the crude extract obtained under different pH and temperature conditions were used to establish the optimal method of extraction. Plant growth parameters such as length, total fresh weight, and chlorophyll composition were evaluated. Additionally, the antibacterial activity of extracts was tested to exclude negative impacts on the beneficial soil microorganisms. The best results were obtained after applying extracts prepared at 25 °C and used at 10% concentration. Enhancement of the tested wheat's growth seems to be related to the composition of the extracts, which we documented as a rich source of macro- and microelements. Our preliminary results are the first confirming the potential of fungal water extracts as factors promoting plant growth. Further detailed investigation needs to be carried out to confirm the effects in real environment conditions. Additionally, the consistency of the plant growth stimulation across different entomopathogenic fungi and agriculturally used plant species should be tested.
RESUMO
Viruses rely on the reprogramming of cellular processes to enable efficient viral replication; this often requires subcompartmentalization within the host cell. Liquid-liquid phase separation (LLPS) has emerged as a fundamental principle to organize and subdivide cellular processes, and plays an important role in viral life cycles. Despite substantial advances in the field, elucidating the exact organization and function of these organelles remains a major challenge. In this review, we summarize the biochemical basis of condensate formation, the role of LLPS during viral infection, and interplay of LLPS with innate immune responses. Finally, we discuss possible strategies and molecules to modulate LLPS during viral infections.
Assuntos
Organelas , Viroses , Humanos , Organelas/química , Organelas/metabolismo , Replicação Viral , Viroses/metabolismoRESUMO
One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Extração Líquido-Líquido/métodos , SARS-CoV-2/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Proteínas Intrinsicamente Desordenadas/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Agregados Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Transcription factor 4 (TCF4) is a class I basic helix-loop-helix (bHLH) transcription factor which regulates the neurogenesis and specialization of cells. TCF4 also plays an important role in the development and functioning of the immune system. Additionally, TCF4 regulates the development of Sertoli cells and pontine nucleus neurons, myogenesis, melanogenesis and epithelial-mesenchymal transition. The ability of transcription factors to fulfil their function often depends on their intracellular trafficking between the nucleus and cytoplasm of the cell. The trafficking is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). We performed research on the TCF4 trafficking regulating sequences by mapping and detailed characterization of motifs potentially acting as the NLS or NES. We demonstrate that the bHLH domain of TCF4 contains an NLS that overlaps two NESs. The results of in silico analyses show high conservation of the sequences, especially in the area of the NLS and NESs. This high conservation is not only between mouse and human TCF4, but also between TCF4 and other mammalian E proteins, indicating the importance of these sequences for the functioning of bHLH class I transcription factors.
Assuntos
Núcleo Celular/metabolismo , Fator de Transcrição 4/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/genética , Humanos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Transporte Proteico , Fator de Transcrição 4/química , Fator de Transcrição 4/genéticaRESUMO
The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptor superfamily, is considered to be functional receptor for the ecdysteroids that coordinate essential biological processes in insects. In this work we have applied a bimolecular fluorescence complementation (BiFC) method to directly analyze the formation of the EcR/Usp complex. The BiFC experiments were carried out in mammalian cells which are routinely used for heterologous studies of the EcR/Usp complex, including experiments on EcR-based artificial molecular gene switches. BiFC analysis, supported by flow cytometry, revealed that EcR-Usp interaction is nuclei-restricted. If expressed separately, Usp and EcR are able to form nuclear complexes in the absence of the cognate dimerization partner. We have observed that Muristerone A that is widely used for the induction of ecdysteroid-dependent transcription in mammalian cells, does not significantly change the number of EcR/Usp and EcR/EcR complexes, and it does not influence their subcellular localization.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Drosophila , Citometria de Fluxo , Fluorescência , Células HeLa , Humanos , Espaço Intracelular , Ligação Proteica , Transporte ProteicoRESUMO
Methoprene tolerant protein (Met) has recently been confirmed as the long-sought juvenile hormone (JH) receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E) and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC) is not homologous to any sequence deposited in the Protein Data Bank (PDB) and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP). The final averaged structure obtained with small angle X-ray scattering (SAXS) experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects.
RESUMO
Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. We performed confocal microscopy analysis of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed us to characterize the details of the subcellular traffic of this protein. We demonstrate that GCE possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Análise de Sequência de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Drosophila melanogaster/efeitos dos fármacos , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Hormônios Juvenis/farmacologia , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Sinais de Exportação Nuclear , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn't been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP-MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn't seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors.